Nucleic acids encoding mutant human CD80 and compositions comprising the same

ABSTRACT

Improved vaccines and methods of using the same are disclosed Immunosuppressive compositions for treating individuals who have autoimmune diseases or transplants and methods of using the same are disclosed.

This application is related to U.S. Provisional Application Ser. No.60/131,764 filed Apr. 30, 1999, which is incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to compositions for and methods ofimmunizing individuals, to immunosuppressive compositions, componentsthereof and methods of making and using the same.

BACKGROUND OF THE INVENTION

CD28 is a cell surface glycoprotein constitutively expressed on mostmature T-cells and thymocytes, while the CTLA-4 receptor is not presenton resting T cells and is only detectable 48 to 72 hours after T cellactivation. The principal ligands for CD28/CTLA-4 molecules are B7.1(CD80) and B7.2 (CD86) expressed on the surface of professional antigenpresenting cells (APC). The biological rationale for the existence of atleast two receptors (CD28 and CTLA-4) and two ligands (CD80 and CD86) isnot clear. It was initially demonstrated that CD80 and CD86 antigenswere functionally similar. However, different roles for theseco-stimulatory molecules were first suggested when the differentpatterns of their expression were determined. CD86 is constitutivelyexpressed on APC and after activation of APC, the expression of CD86 isquickly up-regulated followed by a gradual return to baseline levels.The expression of CD80 is delayed compared to CD86 and its expression ismaximal 48 to 72 hours after the initiation of an immune response.Because CD86 expressed constitutively and up-regulated earlier than CD80it was suggested that CD86 expression is important for the early phaseof an immune response, while CD80 is important for the second.

Functional differences between CD80 and CD86 are further suggested bydata on the binding kinetics of co-stimulatory molecules with CD28 andCTLA-4. Surface plasmon resonance (SPR) analysis has demonstrated thatboth ligands bind to CTLA-4 with higher avidity than to CD28. Furthermeasurements revealed that the CD86/CTLA-4 complex dissociates fasterthan the CD80/CTLA-4 complex. These binding differences combined withthe similar delay in expression of CTLA-4 and CD80 suggest thatfunctional relationship between CTLA-4 and CD80 is probably more potentthan functional relationship between CTLA-4 and CD86 molecules.

Multiple functions for CD80 and CD86 molecules in vitro and in vivo havebeen also reported. Anti-CD86 but not anti-CD80 antibodies block thedevelopment of disease in a mouse model of autoimmune diabetes, whereasthe opposite effect is seen with these antibodies in a murine model ofexperimental allergic encephalomyelitis. Several experimental systemsdemonstrate an important role for CD86 in initiating a T-cell responseto antigen and that the CD80 molecule may play an important role inproviding modulatory signals to these cells. It was observed thatexpression of exogenous human CD86, but not CD80, provides importantactivation signals to murine T cells following DNA vaccination withenvelope proteins from HIV-1. Similar results were observed afterimmunization of mice with DNA encoding HIV-1 or influenza antigens andplasmids encoding murine CD80 and CD86. Thus, functional differencesbetween CD80 and CD86 were not connected with differentialimmunogenicity of human costimulatory molecules expressed in the mouseorganism. It is believed that exogenous human or murine CD86, but notCD80, stimulates anti-viral T-cell activation during DNA immunization.

Vaccines are useful to immunize individuals against target antigens suchas pathogen antigens or antigens associated with cells involved in humandiseases. Antigens associated with cells involved in human diseasesinclude cancer-associated tumor antigens and antigens associated withcells involved in autoimmune diseases.

In designing such vaccines, it has been recognized that vaccines whichproduce the target antigen in the cell of the vaccinated individual areeffective in inducing the cellular arm of the immune system.Specifically, live attenuated vaccines, recombinant vaccines which useavirulent vectors and DNA vaccines all lead to the production ofantigens in the cell of the vaccinated individual which resultsinduction of the cellular arm of the immune system. On the other hand,sub-unit vaccines, which comprise only proteins, and killed orinactivated vaccines induce humoral responses but do not induce goodcellular immune responses.

A cellular immune response is often necessary to provide protectionagainst pathogen infection and to provide effective immune-mediatedtherapy for treatment of pathogen infection, cancer or autoimmunediseases. Accordingly, vaccines which produce the target antigen in thecell of the vaccinated individual such as live attenuated vaccines,recombinant vaccines which use avirulent vectors and DNA vaccines areoften preferred.

While such vaccines are often effective to immunize individualsprophylactically or therapeutically against pathogen infection or humandiseases, there is a need for improved vaccines. There is a need forcompositions and methods which produce an enhanced immune response.

Gene therapy, in contrast to immunization, uses nucleic acid moleculesthat encode non-immunogenic proteins whose expression confers atherapeutic benefit to an individual to whom the nucleic acid moleculesare administered. A specific type of gene therapy relates to thedelivery of genetic material which encodes non-immunogenic proteins thatmodulate immune responses in the individual and thus confer atherapeutic benefit. For example, protocols can be designed to delivergenetic material which encodes non-immunogenic proteins thatdownregulate immune responses associated with an autoimmune disease inan individual and thus confer a therapeutic benefit to the individual.There is a need for compositions and methods which can be used in genetherapy protocols to modulate immune responses.

Modulation of immune responses by alternative means is similarlydesirable to treat diseases such as autoimmune disease andcell/tissue/organ rejection. There is a need for compositions andmethods which can be used to modulate immune responses and to design anddiscover compositions useful to modulate immune responses.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows data from experiments described in the Example showingantigen specific anti-viral CTL responses.

FIGS. 2A and 2B show data from experiments described in the Exampleshowing lymphokine production induced by various constructs.

FIG. 3 shows data from experiments described in the Example showing CTLactivity following administration of constructs.

FIG. 4 shows data from experiments described in the Example showing CTLactivity following administration of constructs measured after theremoval of this population of cells.

FIG. 5 are photographs from experiments described in the Example showinginfiltration of lymphocytes into the muscle of mice immunized withconstructs.

FIG. 6 is a graphic representation of the CD80 molecule.

DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

Applicants have discovered that the C region of human CD80 isresponsible for transmuting a negative signal when an antigen presentingcell (APC) interacts with a T cell. The negative signal results in areduction in the activity of the T cell and thus a reduction in theimmune response generated against the antigen presented by the APC tothe T cell. Specifically, the interaction between a T cell receptor(TCR) on a T cell with an MHC/antigen complex that has been formed on anAPC by the formation of a complex between a major histocompatibilitycomplex (MHC) protein and an antigen is accompanied by the interactionbetween the co-stimulatory molecules CD80 and CD86 present on the APCwith CD28 molecules on the T cell. Such interaction results in T cellactivation and an elevated immune response. However, following T cellactivation, CTLA-4 is expressed by T cells. CTLA-4 interacts with CD80and such interaction results in a dominant negative signal whicheliminates the previous co-stimulatory effect caused by CD80 and CD86interaction with CD28. The result of CD80 interaction with CTLA-4 is areduction in the immune response with which the T cell is involved.

Applicants discovery provides for two distinct aspects of the invention.According to one aspect of the invention, CD80 mutants and nucleic acidsencoding the same are provided which possess the co-stimulatory activityof CD80 but which do not transmute the negative signal associated withCD80 interaction with CTLA-4. Such CD80 mutants are useful inimmunization protocols in which they are delivered, as proteins ornucleic acids encoding such proteins, together with immunogens which aredelivered as protein immunogens or nucleic acid molecules encodingimmunogens. The CD80 mutants of this aspect of the invention aremolecular adjuvants in immunization protocols. According to anotheraspect of the invention, CD80 mutants and nucleic acids encoding thesame are provided which possess the CD80 C region so that they transmutethe negative signal associated with CD80 interaction with CTLA-4. SuchCD80 mutants are useful in the treatment of autoimmune diseases andimmunosuppression protocols associated with cell, tissue and organtransplants. The CD80 mutants which provide the negative signal may bedelivered as proteins or nucleic acids encoding such proteins. The CD80mutants of this aspect of the invention are autoimmune/immunesuppressivetherapeutics.

The nucleotide and amino acid sequences of human CD80 is well known andset forth in Freeman et al. (1989) J. Immunol. 143(8): 2714-2722,Selvakumar et al. (1992) Immunogenetics 36(3): 175-181, Freeman et al.(1991) J. Ex. Med. 174(3): 625-631, Lanier et al. (1989) J. Immunol.154(1): 97-105, and Genbank accession code P33681(www.ncbi.nlm.nih.gov), which are each incorporated herein by reference.

CD86 (B7.2) was first described in Azuma, M. et al. 1993 Nature 366:76-79, which is incorporated herein by reference. FIG. 2B of thatpublication discloses the nucleotide and predicted amino acid sequenceof the B7.2 protein. The sequence information is also available in theGenbank database as U04343 which is incorporated herein by reference.

Human CD80 is expressed as a 288 amino acid protein (1-288) which isprocessed to a mature protein (35-288). CD80 is divided into fourregions: the variable (V) region, the constant (C) region, thetransmembrane region (tm) and the cytoplasmic tail region (ct). Aminoacids 35-242 make up the extracellular domain of the protein. Aminoacids 43-123 make up the V region, also referred to as theImmunoglobulin like V-type domain. Amino acids 155-223 make up the Cregion, also referred to as the Immunoglobulin like C2-type domain.Amino acids 243-263 make up the transmembrane region. Amino acids264-288 make up the cytoplasmic tail.

As used herein, the terms “CD80 mutants”, “C region CD80 mutants”, “Cregion deficient CD80 mutants” and “CD80ΔC mutants” are usedinterchangeably and are meant to refer to molecules which contain eithera functional CD80 or CD86 V region, at least one functional non-C regionof CD80 and which are free of a functional C region of CD80 such that,through the absence of all or part of the C region, such molecules donot transmute the negative signal associated with wild type CD80 Cregion interactions with CTLA-4.

As used herein, references to a “functional region of CD80” as used inthe phrases to “at least one functional non-C region of CD80” and“functional C region of CD80” are meant to refer to complete proteinregions from CD80 as well as partial regions which retain the activityof the complete region. For example, a functional V region of CD80refers to amino acids 43-123 of CD80 or a fragment thereof, includingproteins which include other sequences including but not limited toother CD80 sequences, which retains the ability to bind to CD28. Afunctional C region of CD80 refers to amino acids 155-223 of CD80 or afragment thereof, including proteins which include other sequencesincluding but not limited to other CD80 sequences, which retains theability to bind to CDLA-4 and transmute a negative signal. Thus aprotein free of a functional C region of CD80 may contain a fragment ofamino acids 155-223 of CD80 but such a fragment does not bind to CDLA-4and transmute a negative signal. Similar proteins free of a functional Cregion may include amino acids 155-223 if adjacent sequences are changedto render the C region non-functional through conformational or otherchanges. A functional tm of CD80 refers to amino acids 243-263 of CD80or a fragment thereof which retains the anchor a mutant CD80 proteinwhich comprises it into the cell membrane and thereby prevent secretion.A functional ct of CD80 refers to amino acids 264-288 of CD80 or anyfragment thereof which is retained in the cytoplasm when the mutant CD80protein is expressed.

As used herein, the terms “C region⁺ CD80 proteins” and “C regionproteins” are used interchangeably and are meant to refer to thoseproteins which comprise a functional CD80 C region and due to thepresence of all or part of the C region, such molecules transmute thenegative signal associated with wild type CD80 C region interactionswith CTLA-4.

One aspect of the present invention relates to improved methods andcompositions for vaccination, particularly DNA vaccination in which DNAthat encodes target immunogens is administered into the individual inwhom the DNA is taken up and expressed and an immune response isgenerated against the immunogen. According to aspects of the invention,DNA that encodes a CD80ΔC mutant protein is co-delivered to theindividual and the expression of such DNA produces the CD80ΔC mutantprotein which enhances the immune response induced against theimmunogen.

It has been discovered that the co-production of CD80ΔC mutant proteinin cells of a vaccinated individual that are expressing target antigensresults in an surprisingly enhanced immune response against the targetantigen. By providing an expressible form of nucleotide sequence thatencodes CD80ΔC mutant proteins, vaccines which function by expressingtarget antigen in the cells of the vaccinated individual, such as DNAvaccines, recombinant vector vaccines and attenuated vaccines, thevaccines are improved.

The co-production of CD80ΔC mutant proteins in cells producing antigensresults in enhanced cellular immunity against the antigen. Accordingly,the present invention provides improved vaccines by providing anucleotide sequence that encodes CD80ΔC mutant protein operably linkedto necessary regulatory sequences for expression in vaccinees as part ofvaccines such as DNA vaccines, subunit avirulent recombinant vectorvaccines and live attenuated vaccines. Alternatively, CD80ΔC mutantprotein is delivered as a protein adjuvant together with an immunogen orgene construct encoding an immunogen.

According to some embodiments of the invention in which CD80ΔC mutantsare provided as molecular adjuvants in immunization protocols, theCD80ΔC mutants contain either a functional CD80 V region or a functionalCD86 V region. The CD80ΔC mutants do not contain a functional CD80 Cregion. In some embodiments, the C region is deleted and the V region islinked directly to the transmembrane region. In some embodiments theCD86 C region is inserted in place of the CD80 C region. In someembodiments, non-CD-80, non-CD86 sequences are included in the CD80ΔCmutants after the V region. Some embodiments include the CD80transmembrane region. Some embodiments include the CD86 transmembraneregion. In some embodiments, the CD80 transmembrane region is deletedand not substituted by any other sequences. Some embodiments includenon-CD-80, non-CD86 sequences in place of the CD80 tm. Some embodimentsinclude the CD80 cytoplasmic tail. Some embodiments include the CD86cytoplasmic tail. In some embodiments, the CD80 cytoplasmic tail isdeleted and not substituted by any other sequences. Some embodimentsinclude non-CD-80, non-CD86 sequences in place of the CD80 ct. It hasbeen discovered that in those embodiments in which the CD80ΔC mutantsare delivered to the cells by the administration of genetic materialwhich encodes the CD80ΔC mutant, those CD80ΔC mutants which include atransmembrane region and cytoplasmic tail are particularly effective instimulating immune responses. In some embodiments, the CD80 tm and CD80ct are provided. In some embodiments, the CD86 tm and CD86 ct areprovided. In some embodiments, the CD80 tm and CD86 ct are provided. Insome embodiments, the CD86 tm and CD80 ct are provided. In thoseembodiments in which the CD80ΔC mutants are delivered to the cells bythe administration of CD80ΔC mutant proteins, the CD80ΔC mutant proteinsmay be provided as a soluble protein in which the transmembrane regionand cytoplasmic tail are deleted and, in some cases, replaced with asoluble moiety.

Aspects of the present invention relate to isolated proteins thatcomprises 80V, 80tm and 80ct and is free of 80C; wherein said proteincomprises either 80V or 86 V or both and optionally comprises one ormore of 80tm, 86tm, 80ct and 86ct and wherein:

80V is the variable domain of CD80 or a functional fragment thereof;

86V is the variable domain of CD86 or a functional fragment thereof;

86C is the C domain of CD86 or a functional fragment thereof;

80tm is the transmembrane region of CD80 or a functional fragmentthereof;

86tm is the transmembrane region of CD86 or a functional fragmentthereof;

80ct is the cytoplasmic tail of CD80 or a functional fragment thereof;and

86ct is the cytoplasmic tail of CD86 or a functional fragment thereof.

According to some embodiments, the have the formula:R¹—R²—R³—R⁴—R⁵—R⁶—R⁷—R⁸—R⁹whereinR¹ is 0-50 amino acids;R² is 80V or 86V;R³ is 0-50 amino acids;R⁴ is 86C or 0 amino acids;R⁵ is 0-50 amino acids;R⁶ is 80tm or 86tm;R⁷ is 0-50 amino acids;R⁸ is 80ct or 86ct; andR⁹ is 0-50 amino acids.

In some embodiments R¹ is 0-25 amino acids; R³ is 0-25 amino acids; R⁵is 0-25 amino acids; R⁷ is 0-25 amino acids; and/or R⁹ is 0-25 aminoacids.

In some embodiments R¹ is 0-10 amino acids; R³ is 0-10 amino acids; R⁵is 0-10 amino acids; R⁹ is 0-10 amino acids; and/or R⁹ is 0-10 aminoacids.

In some embodiments, the protein is a CD80 mutant selected from thegroup consisting of:

80V/dele/80tm/80ct;

80V/dele/80tm/86ct;

80V/dele/86tm/80ct;

86V/dele/80tm/80ct;

86V/dele/80tm/86ct;

86V/dele/86tm/80ct;

80V/dele/86tm/86ct;

80V/86C/80tm/80ct;

80V/86C/80tm/86ct;

80V/86C/86tm/80ct;

86V/86C/80tm/80ct;

86V/86C/80tm/86ct;

86V/86C/86tm/80ct;

80V/86C/86tm/86ct;

80V/dele/80tm/dele;

80V/dele/86tm/dele;

86V/dele/80tm/dele;

80V/86C/80tm/dele;

80V/86C/86tm/dele;

86V/86C/80tm/dele;

86V/86C/80tm/dele;

86V/86C/dele/80ct;

80V/86C/dele/80ct;

80V/dele/dele/80ct;

86V/dele/dele/80ct;

80V/86C/dele/dele; and;

80V.

In some embodiments, the CD80 mutant has the formula selected from thegroup consisting of:

R-80V-R-dele-R-80tm-R-80ct-R;

R-80V-R-dele-R-80tm-R-86ct-R;

R-80V-R-dele-R-86tm-R-80ct-R;

R-86V-R-dele-R-80tm-R-80ct-R;

R-86V-R-dele-R-80tm-R-86ct-R;

R-86V-R-dele-R-86tm-R-80ct-R;

R-80V-R-dele-R-86tm-R-86ct-R;

R-80V-R-86C-R-80tm-R-80ct-R;

R-80V-R-86C-R-80tm-R-86ct-R;

R-80V-R-86C-R-86tm-R-80ct-R;

R-86V-R-86C-R-80tm-R-80ct-R;

R-86V-R-86C-R-80tm-R-86ct-R;

R-86V-R-86C-R-86tm-R-80ct-R;

R-80V-R-86C-R-86tm-R-86ct-R;

R-80V-R-dele-R-80tm-R-dele-R;

R-80V-R-dele-R-86tm-R-dele-R;

R-86V-R-dele-R-80tm-R-dele-R;

R-80V-R-86C-R-80tm-R-dele-R;

R-80V-R-86C-R-86tm-R-dele-R;

R-86V-R-86C-R-80tm-R-dele-R;

R-86V-R-86C-R-80tm-R-dele-R;

R-86V-R-86C-R-dele-R-80ct-R;

R-80V-R-86C-R-dele-R-80ct-R;

R-80V-R-dele-R-dele-R-80ct-R;

R-86V-R-dele-R-dele-R-80ct-R;

R-80V-R-86C-R-dele-R-dele-R; and

R-80V-R; wherein

80V is the variable domain of CD80 or a functional fragment thereof;

86V is the variable domain of CD86 or a functional fragment thereof;

86C is the C domain of CD86 or a functional fragment thereof;

80tm is the transmembrane region of CD86 or a functional fragmentthereof;

86tm is the transmembrane region of CD86 or a functional fragmentthereof;

80ct is the cytoplasmic trio of CD86 or a functional fragment thereof;

86ct is the cytoplasmic tail of CD86 or a functional fragment thereof;

dele is 0 amino acids; and

R are each independently 0-100 amino acids.

In some embodiments, R are each independently 0-50 amino acids.

In some embodiments, R are each independently 0-30 amino acids.

In some embodiments, R are each independently 0-20 amino acids.

In some embodiments of the invention, the CD80 mutant is selected fromthe group consisting of:

CD80 with the C domain deleted;

CD80 with the C domain deleted and a CD86 transmembrane regionsubstituting the CD80 transmembrane region;

CD80 with the C domain deleted and a CD86 cytoplasmic tail regionsubstituting the CD80 cytoplasmic tail region;

CD80 with the C domain deleted and a CD86 V domain substituting the CD80V domain;

CD80 with the C domain deleted and a CD86 V domain substituting the CD80V domain and a CD86 transmembrane region substituting the CD80transmembrane region;

CD80 with the C domain deleted and a CD86 V domain substituting the CD80V domain and a CD86 cytoplasmic tail region substituting the CD80cytoplasmic tail region;

CD80 with the C domain deleted and a CD86 transmembrane regionsubstituting the CD80 transmembrane region and a CD86 cytoplasmic tailregion substituting the CD80 cytoplasmic tail region;

CD80 with a CD86 C domain substituting the CD80 C domain;

CD80 with a CD86 C domain substituting the CD80 C domain and a CD86transmembrane region substituting the CD80 transmembrane region;

CD80 with a CD86 C domain substituting the CD80 C domain and a CD86cytoplasmic tail region substituting the CD80 cytoplasmic tail region;

CD80 with a CD86 C domain substituting the CD80 C domain and a CD86 Vdomain substituting the CD80 V domain;

CD80 with a CD86 C domain substituting the CD80 C. domain and a CD86 Vdomain substituting the CD80 V domain and a CD86 transmembrane regionsubstituting the CD80 transmembrane region;

CD80 with a CD86 C domain substituting the CD80 C domain and a CD86 Vdomain substituting the CD80 V domain and a CD86 cytoplasmic tail regionsubstituting the CD80 cytoplasmic tail region;

CD80 with a CD86 C domain substituting the CD80 C domain and a CD86transmembrane region substituting the CD80 transmembrane region and aCD86 cytoplasmic tail region substituting the CD80 cytoplasmic tailregion;

CD80 with the C domain deleted and the cytoplasmic tail region deleted;

CD80 with the C domain deleted and the cytoplasmic tail region deletedand a CD86 transmembrane region substituting the CD80 transmembraneregion;

CD80 with the C domain deleted and the cytoplasmic tail region deletedand a CD86 V domain substituting the CD80 V domain;

CD80 with a CD86 C domain substituting the CD80 C. domain and a CD86transmembrane region substituting the CD80 transmembrane region and aCD86 cytoplasmic tail region substituting the CD80 cytoplasmic tailregion;

CD80 with a CD86 C domain substituting the CD80 C domain and thecytoplasmic tail region deleted;

CD80 with a CD86 C domain substituting the CD80 C domain and thecytoplasmic tail region deleted and a CD86 transmembrane regionsubstituting the CD80 transmembrane region;

CD80 with a CD86 V domain substituting the CD80 V domain and a CD86 Cdomain substituting the CD80 C. domain and the cytoplasmic tail regiondeleted;

CD80 with a CD86 V domain substituting the CD80 V domain and a CD86 Cdomain substituting the CD80 C domain and the transmembrane regiondeleted;

CD80 with a CD86 C domain substituting the CD80 C domain and thetransmembrane region deleted;

CD80 with the C domain deleted and the transmembrane region deleted;

CD80 with the C domain deleted and CD86 V domain substituting the CD80 Vdomain and the transmembrane region deleted;

CD80 with a CD86 C domain substituting the CD80 C domain and thetransmembrane region deleted and the cytoplasmic tail region deleted;

CD80 with the domain deleted, the transmembrane region deleted and thecytoplasmic tail region deleted; and

the CD80 variable domain or functional fragments thereof.

Protein forms of the CD80ΔC mutants can be formulated as components invaccines or genetic constructs which include coding sequences thatencode the CD80ΔC mutants may be provided as components of vaccines. Ineither case, such vaccines may be used in prophylactic or therapeuticmethods.

According to some preferred embodiments of the invention, DNA vaccinesare provided which contain DNA molecules that contain coding sequencesencoding an immunogen and a CD80ΔC mutant. An improvement of the presentinvention relates to the inclusion of genetic material for theco-production of a CD80ΔC mutant protein in addition to the productionof the antigenic target encoded by nucleic acid sequences of the DNAvaccines.

The present invention relates to methods of introducing genetic materialinto the cells of an individual in order to induce immune responsesagainst proteins and peptides which are encoded by the genetic material.The methods comprise the steps of administering to the tissue of saidindividual, either a single nucleic acid molecule that comprises anucleotide sequence that encodes a target protein and a nucleotidesequence that encodes a CD80ΔC mutant protein, or a composition havingtwo nucleic acid molecules, one that comprises a nucleotide sequencethat encodes a target protein and one that comprises a nucleotidesequence that encodes a CD80ΔC mutant protein. The nucleic acidmolecule(s) may be provided as plasmid DNA, the nucleic acid moleculesof recombinant vectors or as part of the genetic material provided in anattenuated vaccine.

According to the present invention, compositions and methods areprovided which prophylactically and/or therapeutically immunize anindividual against a pathogen or abnormal, disease-related cell. Thegenetic material that encodes a target protein, i.e. a peptide orprotein that shares at least an epitope with an immunogenic proteinfound on the pathogen or cells to be targeted, and genetic material thatencodes a CD80ΔC mutant protein. The genetic material is expressed bythe individual's cells and serves as an immunogenic target against whichan immune response is elicited. The resulting immune response reactswith a pathogen or cells to be targeted and is broad based: in additionto a humoral immune response, both arms of the cellular immune responseare elicited. The methods of the present invention are useful forconferring prophylactic and therapeutic immunity. Thus, a method ofimmunizing includes both methods of protecting an individual frompathogen challenge, or occurrence or proliferation of specific cells aswell as methods of treating an individual suffering from pathogeninfection, hyperproliferative disease or autoimmune disease.

As used herein the terms “target protein” and “immunogen” are usedinterchangeably and are meant to refer to peptides and protein encodedby gene constructs which act as protein targets for an immune response.The target protein is a protein against which an immune response can beelicited. The target protein is an immunogenic protein which shares atleast an epitope with a protein from the pathogen or undesirablecell-type, such as a cancer cell or a cell involved in autoimmunedisease, against which immunization is required. The immune responsedirected against the target protein will protect the individual againstand treat the individual for the specific infection or disease withwhich the target protein is associated. The target protein does not needto be identical to the protein against which an immune response isdesired. Rather, the target protein must be capable of inducing animmune response that cross reacts to the protein against which theimmune response is desired.

The present invention is useful to elicit broad immune responses againsta target protein, i.e. proteins specifically associated with pathogensor the individual's own “abnormal” cells. The present invention isuseful to immunize individuals against pathogenic agents and organismssuch that an immune response against a pathogen protein providesprotective immunity against the pathogen. The present invention isuseful to combat hyperproliferative diseases and disorders such ascancer by eliciting an immune response against a target protein that isspecifically associated with the hyperproliferative cells. The presentinvention is useful to combat autoimmune diseases and disorders byeliciting an immune response against a target protein that isspecifically associated with cells involved in the autoimmune condition.

According to the present invention, DNA or RNA that encodes a targetprotein and a CD80ΔC mutant protein is introduced into the cells oftissue of an individual where it is expressed, thus producing the targetprotein and the CD80ΔC mutant protein. The DNA or RNA sequences encodingthe target protein and the CD80ΔC mutant are each linked to regulatoryelements necessary for expression in the cells of the individual.Regulatory elements for DNA expression include a promoter and apolyadenylation signal. In addition, other elements, such as a Kozakregion, may also be included in the genetic construct. The preferredembodiments include nucleotide sequences encoding the target protein andCD80ΔC mutant protein provided as separate expressible forms in whicheach of the target protein and CD80ΔC mutant protein is linked to itsown set of regulatory elements necessary for expression in the cell ofthe individual. However, the present invention additional relates toembodiments in which the target protein and CD80ΔC mutant protein areprovided as a single genetic construct. In some such embodiment, thepolyprotein which is produced by the single expressible form may beprocessed into two separate proteins or it may exist as a chimericprotein which functions both as the target protein and CD80ΔC mutant. Insome embodiments, nucleic acid sequences encoding two or more copies ofthe target protein and/or two or more copies CD80ΔC mutant protein maybe provided in a single expressible form of a gene construct.Polyproteins encoded therein may be processed into subunits followingexpression or maintained as functional polyproteins.

As used herein, the term “expressible form” refers to gene constructswhich contain the necessary regulatory elements operable linked to acoding sequence that encodes a target protein and/or CD80ΔC mutantprotein, such that when present in the cell of the individual, thecoding sequence will be expressed.

As used herein, the term “sharing an epitope” refers to proteins whichcomprise at least one epitope that is identical to or substantiallysimilar to an epitope of another protein.

As used herein, the term “substantially similar epitope” is meant torefer to an epitope that has a structure which is not identical to anepitope of a protein but nonetheless invokes an cellular or humoralimmune response which cross reacts to that protein.

Genetic constructs comprise a nucleotide sequence that encodes a targetprotein and/or a CD80ΔC mutant protein operably linked to regulatoryelements needed for gene expression. According to the invention,combinations of gene constructs which include one that comprises anexpressible form of the nucleotide sequence that encodes a targetprotein and one that includes an expressible form of the nucleotidesequence that encodes a CD80ΔC mutant protein are provided.Incorporation into a living cell of the DNA or RNA molecule(s) whichinclude the combination of gene constructs results in the expression ofthe DNA or RNA and production of the target protein and a CD80ΔC mutantprotein. A surprisingly enhanced immune response against the targetprotein results.

The present invention may be used to immunize an individual against allpathogens such as viruses, prokaryote and pathogenic eukaryoticorganisms such as unicellular pathogenic organisms and multicellularparasites. The present invention is particularly useful to immunize anindividual against those pathogens which infect cells and which are notencapsulated such as viruses, and prokaryote such as gonorrhea, listeriaand shigella. In addition, the present invention is also useful toimmunize an individual against protozoan pathogens which include a stagein the life cycle where they are intracellular pathogens. As usedherein, the term “intracellular pathogen” is meant to refer to a virusor pathogenic organism that, at least part of its reproductive or lifecycle, exists within a host cell and therein produces or causes to beproduced, pathogen proteins. Table 1 provides a listing of some of theviral families and genera for which vaccines according to the presentinvention can be made. DNA constructs that comprise DNA sequences whichencode the peptides that comprise at least an epitope identical orsubstantially similar to an epitope displayed on a pathogen antigen suchas those antigens listed on the tables are useful in vaccines. Moreover,the present invention is also useful to immunize an individual againstother pathogens including prokaryotic and eukaryotic protozoan pathogensas well as multicellular parasites such as those listed on Table 2.

In order to produce a genetic vaccine to protect against pathogeninfection, genetic material which encodes immunogenic proteins againstwhich a protective immune response can be mounted must be included in agenetic construct as the coding sequence for the target. Whether thepathogen infects intracellularly, for which the present invention isparticularly useful, or extracellularly, it is unlikely that allpathogen antigens will elicit a protective response. Because DNA and RNAare both relatively small and can be produced relatively easily, thepresent invention provides the additional advantage of allowing forvaccination with multiple pathogen antigens. The genetic construct usedin the genetic vaccine can include genetic material which encodes manypathogen antigens. For example, several viral genes may be included in asingle construct thereby providing multiple targets.

Tables 1 and 2 include lists of some of the pathogenic agents andorganisms for which genetic vaccines can be prepared to protect anindividual from infection by them. In some preferred embodiments, themethods of immunizing an individual against a pathogen are directedagainst HIV, HTLV or HBV.

Another aspect of the present invention provides a method of conferringa broad based protective immune response against hyperproliferatingcells that are characteristic in hyperproliferative diseases and to amethod of treating individuals suffering from hyperproliferativediseases. As used herein, the term “hyperproliferative diseases” ismeant to refer to those diseases and disorders characterized byhyperproliferation of cells Examples of hyperproliferative diseasesinclude all forms of cancer and psoriasis.

It has been discovered that introduction of a genetic construct thatincludes a nucleotide sequence which encodes an immunogenic“hyperproliferating cell”-associated protein into the cells of anindividual results in the production of those proteins in the vaccinatedcells of an individual. As used herein, the term“hyperproliferative-associated protein” is meant to refer to proteinsthat are associated with a hyperproliferative disease. To immunizeagainst hyperproliferative diseases, a genetic construct that includes anucleotide sequence which encodes a protein that is associated with ahyperproliferative disease is administered to an individual.

In order for the hyperproliferative-associated protein to be aneffective immunogenic target, it must be a protein that is producedexclusively or at higher levels in hyperproliferative cells as comparedto normal cells. Target antigens include such proteins, fragmentsthereof and peptides which comprise at least an epitope found on suchproteins. In some cases, a hyperproliferative-associated protein is theproduct of a mutation of a gene that encodes a protein. The mutated geneencodes a protein which is nearly identical to the normal protein exceptit has a slightly different amino acid sequence which results in adifferent epitope not found on the normal protein. Such target proteinsinclude those which are proteins encoded by oncogenes such as myb, myc,fyn, and the translocation gene bcr/abl, ras, src, P53, neu, trk andEGRF. In addition to oncogene products as target antigens, targetproteins for anti-cancer treatments and protective regimens includevariable regions of antibodies made by B cell lymphomas and variableregions of T cell receptors of T cell lymphomas which, in someembodiments, are also used target antigens for autoimmune disease. Othertumor-associated proteins can be used as target proteins such asproteins which are found at higher levels in tumor cells including theprotein recognized by monoclonal antibody 17-1A and folate bindingproteins.

While the present invention may be used to immunize an individualagainst one or more of several forms of cancer, the present invention isparticularly useful to prophylactically immunize an individual who ispredisposed to develop a particular cancer or who has had cancer and istherefore susceptible to a relapse. Developments in genetics andtechnology as well as epidemiology allow for the determination ofprobability and risk assessment for the development of cancer inindividual. Using genetic screening and or family health histories, itis possible to predict the probability a particular individual has fordeveloping any one of several types of cancer.

Similarly, those individuals who have already developed cancer and whohave been treated to remove the cancer or are otherwise in remission areparticularly susceptible to relapse and reoccurrence. As part of atreatment regimen, such individuals can be immunized against the cancerthat they have been diagnosed as having had in order to combat arecurrence. Thus, once it is known that an individual has had a type ofcancer and is at risk of a relapse, they can be immunized in order toprepare their immune system to combat any future appearance of thecancer.

The present invention provides a method of treating individualssuffering from hyperproliferative diseases. In such methods, theintroduction of genetic constructs serves as an immunotherapeutic,directing and promoting the immune system of the individual to combathyperproliferative cells that produce the target protein.

The present invention provides a method of treating individualssuffering from autoimmune diseases and disorders by conferring a broadbased protective immune response against targets that are associatedwith autoimmunity including cell receptors and cells which produce“self”-directed antibodies.

T cell mediated autoimmune diseases include Rheumatoid arthritis (RA),multiple sclerosis (MS), Sjogren's syndrome, sarcoidosis, insulindependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactivearthritis, ankylosing spondylitis, scieroderma, polymyositis,dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis,Crohn's disease and ulcerative colitis. Each of these diseases ischaracterized by T cell receptors that bind to endogenous antigens andinitiate the inflammatory cascade associated with autoimmune diseases.Vaccination against the variable region of the T cells would elicit animmune response including CTLs to eliminate those T cells.

In RA, several specific variable regions of T cell receptors (TCRs)which are involved in the disease have been characterized. These TCRsinclude Vβ-3, Vβ-14, Vβ-17 and Vα-17. Thus, vaccination with a DNAconstruct that encodes at least one of these proteins will elicit animmune response that will target T cells involved in RA. See: Howell, M.D., et al., 1991 Proc. Natl. Acad. Sci. USA 88: 10921-10925; Paliard,X., et al, 1991 Science 253: 325-329; Williams, W. V., et al. 1992 J.Clin. Invest. 90: 326-333; each of which is incorporated herein byreference.

In MS, several specific variable regions of TCRs which are involved inthe disease have been characterized. These TCRs include Vβ-7 and Vα-10.Thus, vaccination with a DNA construct that encodes at least one ofthese proteins will elicit an immune response that will target T cellsinvolved in MS. See: Wucherpfennig, K. W., et al., 1990 Science 248:1016-1019; Oksenberg, J. R., et al, 1990 Nature 345: 344-346; each ofwhich is incorporated herein by reference.

In scleroderma, several specific variable regions of TCRs which areinvolved in the disease have been characterized. These TCRs includeVβ-6, Vβ-8, Vβ-14 and Vα-16, Vα-3C, Vα-7, Vα-14, Vα-15, Vα-16, Vα-28 andVα-12. Thus, vaccination with a DNA construct that encodes at least oneof these proteins will elicit an immune response that will target Tcells involved in scleroderma.

In order to treat patients suffering from a T cell mediated autoimmunedisease, particularly those for which the variable region of the TCR hasyet to be characterized, a synovial biopsy can be performed. Samples ofthe T cells present can be taken and the variable region of those TCRsidentified using standard techniques. Genetic vaccines can be preparedusing this information.

B cell mediated autoimmune diseases include Lupus (SLE), Grave'sdisease, myasthenia gravis, autoimmune hemolytic anemia, autoimmunethrombocytopenia, asthma, cryoglobulinemia, primary biliary sclerosisand pernicious anemia. Each of these diseases is characterized byantibodies which bind to endogenous antigens and initiate theinflammatory cascade associated with autoimmune diseases. Vaccinationagainst the variable region of antibodies would elicit an immuneresponse including CTLs to eliminate those B cells that produce theantibody.

In order to treat patients suffering from a B cell mediated autoimmunedisease, the variable region of the antibodies involved in theautoimmune activity must be identified. A biopsy can be performed andsamples of the antibodies present at a site of inflammation can betaken. The variable region of those antibodies can be identified usingstandard techniques. Genetic vaccines can be prepared using thisinformation.

In the case of SLE, one antigen is believed to be DNA. Thus, in patientsto be immunized against SLE, their sera can be screened for anti-DNAantibodies and a vaccine can be prepared which includes DNA constructsthat encode the variable region of such anti-DNA antibodies found in thesera.

Common structural features among the variable regions of both TCRs andantibodies are well known. The DNA sequence encoding a particular TCR orantibody can generally be found following well known methods such asthose described in Kabat, et al. 1987 Sequence of Proteins ofImmunological Interest U.S. Department of Health and Human Services,Bethesda Md., which is incorporated herein by reference. In addition, ageneral method for cloning functional variable regions from antibodiescan be found in Chaudhary, V. K., et al., 1990 Proc. Natl. Acad. Sci.USA 87: 1066, which is incorporated herein by reference.

The present invention provides an improved method of immunizingindividuals that comprises the step of delivering gene constructs to thecells of individuals as part of vaccine compositions which include areprovided which include DNA vaccines, attenuated live vaccines andrecombinant vaccines. The gene constructs comprise a nucleotide sequencethat encodes an immunomodulating protein and that is operably linked toregulatory sequences that can function in the vaccinee to effectexpression. The improved vaccines result in an enhanced cellular immuneresponse.

In some methods of immunizing, the individual is administered a geneconstruct encoding an immunogen and a genetic construct encoding aCD80ΔC mutant protein. In some methods of immunizing, the individual isadministered a gene construct encoding both an immunogen and a CD80ΔCmutant protein. In some alternative methods of immunizing, theindividual is administered an immunogen and a CD80ΔC mutant protein. Insome alternative methods of immunizing, the individual is administered aprotein immunogen and genetic construct encoding a CD80ΔC mutantprotein. In some alternative methods of immunizing, the individual isadministered a gene construct encoding an immunogen and a CD80ΔC mutantprotein.

According to another aspect of the invention, CD80 C region proteins areprovided to suppress immune responses associated with autoimmunediseases and transplant rejections. The CD80 C. region proteins containa functional CD80 C. region. Functional fragments of the CD80 C regioncan be identified routinely. In some embodiments, functional fragmentsof the CD80 C region are less than 60 amino acids. In some embodiments,functional fragments of the CD80 C region are less than 50 amino acids.In some embodiments, functional fragments of the CD80 C region are lessthan 40 amino acids. In some embodiments, functional fragments of theCD80 C region are less than 30 amino acids. In some embodiments,functional fragments of the CD80 C region are less than 20 amino acids.In some embodiments, functional fragments of the CD80 C region are lessthan 15 amino acids. In some embodiments, functional fragments of theCD80 C region are less than 10 amino acids.

In some embodiments, the V region is deleted. In some embodiments, theCD80 or CD86 V region is present. Some embodiments include the CD80transmembrane region. Some embodiments include the CD86 transmembraneregion. In some embodiments, the CD80 transmembrane region is deletedand not substituted by any other sequences. Some embodiments includenon-CD-80, non-CD86 sequences. Some embodiments include non-CD-80,non-CD86 sequences in place of the CD80tm. Some embodiments include theCD80 cytoplasmic tail. Some embodiments include the CD86 cytoplasmictail. In some embodiments, the CD80 cytoplasmic tail is deleted and notsubstituted by any other sequences. Some embodiments include non-CD-80,non-CD86 sequences in place of the CD80 ct.

According to some embodiments, the non-CD80 protein comprises at leastthe C domain of CD80 or a functional fragment thereof. As used herein,the term non-CD80 protein is meant to refer to a protein which differsfrom wildtype CD80 but comprises a CD80 C domain or a functionalfragment thereof. In some embodiments, the non-CD80 protein have theformula:R¹—R²—R³—R⁴—R⁵—R⁶—R⁷—R⁸—R⁹whereinR¹ is 0-50 amino acids;R² is 80V or 86V;R³ is 0-50 amino acids;R⁴ is 80C;R⁵ is 0-50 amino acids;R⁶ is 80tm or 86tm;R⁷ is 0-50 amino acids;R⁸ is 80ct or 86ct; andR⁹ is 0-50 amino acidswherein

-   -   80V is the variable domain of CD80 or a functional fragment        thereof;    -   86V is the variable domain of CD86 or a functional fragment        thereof;    -   80C is the C domain of CD80 or a functional fragment thereof;    -   80tm is the transmembrane region of CD80 or a functional        fragment thereof;    -   86tm is the transmembrane region of CD86 or a functional        fragment thereof;    -   80ct is the cytoplasmic tail of CD80 or a functional fragment        thereof; and    -   86ct is the cytoplasmic tail of CD86 or a functional fragment        thereof.

According to some embodiments of the invention, the isolated non-CD80protein that comprises at least the C domain of CD80 or a functionalfragment thereof has the formula selected from the group consisting of:

R-dele-R-80C-R-80tm-R-80ct-R;

R-dele-R-80C-R-80tm-R-dele-R;

R-80V-R-80C-R-80tm-R-dele-R;

R-80V-R-80C-R-dele-R-dele-R;

R-86V-R-80C-R-80tm-R-80ct-R;

R-86V-R-80C-R-80tm-R-dele-R;

R-86V-R-80C-R-dele-R-dele-R;

R-80V-R-80C-R-86tm-R-80ct-R;

R-dele-R-80C-R-86tm-R-80ct-R;

R-dele-R-80C-R-86tm-R-dele-R;

R-80V-R-80C-R-86tm-R-dele-R;

R-80V-R-80C-R-80tm-R-86ct-R;

R-dele-R-80C-R-80tm-R-86ct-R;

R-86V-R-80C-R-86tm-R-80ct-R;

R-86V-R-80C-R-80tm-R-86ct-R;

R-86V-R-80C-R-86tm-R-dele-R;

R-dele-R-80C-R-86tm-R-86ct-R; and

R-86V-R-80C-R-86tm-R-86ct;

wherein

-   -   80V is the variable domain of CD80 or a functional fragment        thereof;    -   86V is the variable domain of CD86 or a functional fragment        thereof;    -   80C is the C domain of CD80 or a functional fragment thereof;    -   80tm is the transmembrane region of CD80 or a functional        fragment thereof;    -   86tm is the transmembrane region of CD86 or a functional        fragment thereof;    -   80ct is the cytoplasmic tail of CD80 or a functional fragment        thereof;    -   86ct is the cytoplasmic tail of CD86 or a functional fragment        thereof;    -   dele is 0 amino acids; and    -   each R is each independently 0-100 amino acids.        In some embodiments, each R is independently 0-50 amino acids;        in some embodiments, each R is independently 0-30 amino acids;        in some embodiments, each R is independently 0-20 amino acids.

In some embodiments of the invention, the non-CD80 protein is selectedfrom the group consisting of:

a mutant CD80 with the variable domain deleted;

a mutant CD80 with the variable domain deleted and the cytoplasmic taildeleted;

a mutant CD80 with the cytoplasmic tail deleted;

a mutant CD80 with the transmembrane region deleted and the cytoplasmictail deleted;

a mutant CD80 with a CD86 variable domain substituted in place of theCD80 variable domain;

a mutant CD80 with a CD86 variable domain substituted in place of theCD80 variable domain and the cytoplasmic tail deleted;

a mutant CD80 with a CD86 variable domain substituted in place of theCD80 variable domain and the transmembrane region deleted and thecytoplasmic tail deleted;

a mutant CD80 with a CD86 transmembrane region substituted in place ofthe CD80 transmembrane region;

a mutant CD80 with the variable region deleted and a CD86 transmembraneregion substituted in place of the CD80 transmembrane region;

a mutant CD80 with the variable region deleted, the cytoplasmic taildeleted and a CD86 transmembrane region substituted in place of the CD80transmembrane region;

a mutant CD80 with the cytoplasmic tail deleted and a CD86 transmembraneregion substituted in place of the CD80 transmembrane region;

a mutant CD80 with a CD86 cytoplasmic tail substituted in place of theCD80 cytoplasmic tail;

a mutant CD80 with the variable region deleted and a CD86 cytoplasmictail substituted in place of the CD80 cytoplasmic tail;

a mutant CD80 with a CD86 variable domain substituted in place of theCD80 variable domain and a CD86 transmembrane region substituted inplace of the CD80 transmembrane region;

a mutant CD80 with a CD86 variable domain substituted in place of theCD80 variable domain and a CD86 cytoplasmic tail substituted in place ofthe CD80 cytoplasmic tail;

a mutant CD80 with a CD86 variable domain substituted in place of theCD80 variable domain and a CD86 transmembrane region substituted inplace of the CD80 transmembrane region and the cytoplasmic tail deleted;

a mutant CD80 with the variable domain deleted and a CD86 transmembraneregion substituted in place of the CD80 transmembrane region and CD86cytoplasmic tail substituted in place of the CD80 cytoplasmic tail; and

a mutant CD80 with a CD86 variable domain substituted in place of theCD80 variable domain and a CD86 transmembrane region substituted inplace of the CD80 transmembrane region and CD86 cvtoplasmic tailsubstituted in place of the CD80 cytoplasmic tail.

The CD80 C region proteins are provided as either proteins or geneticconstructs that encode CD80 C region. Delivery of genetic constructswhich comprise coding sequences that encode the wild type CD80,dele/80C/80tm/80ct, dele/80C/80tm/86ct dele/80C/86tm/80ct ordele/80C/86tm/86ct provide particularly effective results inimmunosuppression and treatment of autoimmune diseases.

The methods of this aspect of the invention are useful to treatautoimmune diseases and disorders. Those skilled in the art can identifyindividuals who have autoimmune diseases or disorders. Examples ofautoimmune diseases and disorders include T cell mediated autoimmunediseases such as rheumatoid arthritis (RA), multiple sclerosis (MS),Sjogren's syndrome, sarcoidosis, insulin dependent diabetes mellitus(IDDM), autoimmune thyroiditis, reactive arthritis, ankylosingspondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis,vasculitis, Wegener's granulomatosis, Crohn's disease and ulcerativecolitis, and B cell mediated autoimmune diseases such as Lupus (SLE),Grave's disease, myasthenia gravis, autoimmune hemolytic anemia,autoimmune thrombocytopenia, asthma, cryoglobulinemia, primary biliarysclerosis and pernicious anemia.

The methods of this aspect of the invention are also useful to suppressimmune responses in individuals undergoing transplant proceduresincluding cell transplants such as bone marrow and brain cell grafts,tissue transplants such as cornea and skin grafts and myoplastyprocedures, and organ transplants such as liver, lung, kidney and heart.

The methods of making and delivering compositions of the presentinvention are generally the same for immunization protocols as well asnon-immunogenic therapeutic protocols.

As used herein, the term “protein” is meant to include proteinaceousmolecules including peptides, polypeptides and proteins. Someembodiments of the invention relate to delivery of proteins through theadministration of nucleic acids, particularly DNA, and to methods ofusing the same. For example, in some methods of immunizing, nucleicacids that encode immunogenic proteins and CD80ΔC mutant proteins areadministered to individuals. Likewise, in some methods oftreatingautoimmune diseases and preventing graft/transplant rejections byimmunosuppression, nucleic acids that encode CD80 C region proteins areadministered to individuals. As used herein, the term “gene constructsof the invention” is intended to mean gene constructs that includecoding sequences which encode immunogenic proteins, CD80ΔC mutantproteins and CD80 C region proteins which each can be produced bysimilar means and which can be formulated and administered in a similarmanner for use in methods of the invention.

DNA vaccines are described in U.S. Pat. No. 5,593,972, U.S. Pat. No.5,589,466, PCT/US90/01515, PCT/US93/02338, PCT/US93/048131, andPCT/US94/00899, which are each incorporated herein by reference. Inaddition to the delivery protocols described in those applications,alternative methods of delivering DNA are described in U.S. Pat. Nos.4,945,050 and 5,036,006, which are both incorporated herein byreference. DNA vaccine protocols are useful to immunize individuals. Theteachings can be applied in the present invention to aspects in whichindividuals with autoimmune disease and transplant rejections aretreated using gene constructs that encode CD80 C region proteins. Insuch embodiments, no coding sequences encoding immunogens are provided.

When taken up by a cell, the genetic constructs of the invention mayremain present in the cell as a functioning extrachromosomal moleculeand/or integrate into the cell's chromosomal DNA. DNA may be introducedinto cells where it remains as separate genetic material in the form ofa plasmid or plasmids. Alternatively, linear DNA which can integrateinto the chromosome may be introduced into the cell. When introducingDNA into the cell, reagents which promote DNA integration intochromosomes may be added. DNA sequences which are useful to promoteintegration may also be included in the DNA molecule. Alternatively, RNAmay be administered to the cell. It is also contemplated to provide thegenetic constructs of the invention as a linear minichromosome includinga centromere, telomeres and an origin of replication.

Genetic constructs of the invention include regulatory elementsnecessary for gene expression of a nucleic acid molecule. The elementsinclude: a promoter, an initiation codon, a stop codon, and apolyadenylation signal. In addition, enhancers are often required forgene expression of the sequence that encodes the protein of theinvention. It is necessary that these elements be operable linked to thesequence that encodes the desired proteins and that the regulatoryelements are operably in the individual to whom they are administered.

Initiation codons and stop codon are generally considered to be part ofa nucleotide sequence that encodes the desired protein. However, it isnecessary that these elements are functional in the individual to whomthe gene construct is administered. The initiation and terminationcodons must be in frame with the coding sequence.

Promoters and polyadenylation signals used must be functional within thecells of the individual.

Examples of promoters useful to practice the present invention,especially in the production of a genetic vaccine for humans, includebut are not limited to promoters from Simian Virus 40 (SV40), MouseMammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV)such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV,Cytomegalovirus (CMV) such as the CMV immediate early promoter, EpsteinBarr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters fromhuman genes such as human Actin, human Myosin, human Hemoglobin, humanmuscle creatine and human metalothionein.

Examples of polyadenylation signals useful to practice the presentinvention, especially in the production of a genetic vaccine for humans,include but are not limited to human and bovine growth hormonepolyadenylation signals, SV40 polyadenylation signals and LTRpolyadenylation signals. In particular, the SV40 polyadenylation signalwhich is in pCEP4 plasmid (Invitrogen, San Diego Calif.), referred to asthe SV40 polyadenylation signal, is used.

In addition to the regulatory elements required for DNA expression,other elements may also be included in the DNA molecule. Such additionalelements include enhancers. The enhancer may be selected from the groupincluding but not limited to human Actin, human Myosin, humanHemoglobin, human muscle creatine and viral enhancers such as those fromCMV, RSV and EBV.

Genetic constructs of the invention can be provided with mammalianorigin of replication in order to maintain the constructextrachromosomally and produce multiple copies of the construct in thecell. Plasmids pCEP4 and pREP4 from Invitrogen (San Diego, Calif.)contain the Epstein Barr virus origin ofreplication and nuclear antigenEBNA-1 coding region which produces high copy episomal replicationwithout integration.

In some preferred embodiments related to immunization applications,nucleic acid molecule(s) are delivered which include nucleotidesequences that encode a target protein, a CD80ΔC mutant protein and,additionally, genes for proteins which further enhance the immuneresponse against such target proteins. Examples of such genes are thosewhich encode cytokines and lymphokines such as α-interferon,gamma-interferon, platelet derived growth factor (PDGF), GC-SF, GM-CSF,TNF, epidermal growth factor (EGF), IL-1, IL-2, IL4, IL-6, IL-8, IL-10and IL-12.

In order to maximize protein production, regulatory sequences may beselected which are well suited for gene expression in the cells theconstruct is administered into. Moreover, codons may be selected whichare most efficiently transcribed in the cell. One having ordinary skillin the art can produce DNA constructs which are functional in the cells.

The methods of the present invention, whether methods of immunizing ormethods of immunosuppressing, comprise the step of administering nucleicacid molecules to tissue of the individual. In some preferredembodiments, the nucleic acid molecules are administeredintramuscularly, intranasally, intraperatoneally, subcutaneously,intradermally, intravenously, by aerosol administration to lung tissueor topically or by lavage to mucosal tissue selected from the groupconsisting of vaginal, rectal, urethral, buccal and sublingual.

An aspect of the present invention relates to pharmaceuticalcompositions useful in the methods of the present invention. Thepharmaceutical compositions comprise a nucleic acid molecule, preferablya DNA molecule comprising a nucleotide sequence that encodes one or moreproteins operably linked to regulatory elements necessary for expressionin the cells of the individual. The pharmaceutical compositions furthercomprise a pharmaceutically acceptable carrier or diluent. The term“pharmaceutical” is well known and widely understood by those skilled inthe art. As used herein, the terms “pharmaceutical compositions” and“injectable pharmaceutical compositions” are meant to have theirordinary meaning as understood by those skilled in the art.Pharmaceutical compositions are required to meet specific standardsregarding sterility, pyrogens, particulate matter as well as isotonicityand pH. For example, injectable pharmaceuticals are sterile and pyrogenfree.

Pharmaceutical compositions according to the present invention maycomprise about 1 ng to about 10,000 μg of DNA. In some preferredembodiments, the pharmaceutical compositions contain about 2000 μg, 3000μg, 4000 μg or 5000 μg of DNA. In some preferred embodiments, thepharmaceutical compositions contain about 1000 μg of DNA. In somepreferred embodiments, the pharmaceutical compositions contain about 10ng to about 800 μg of DNA. In some preferred embodiments, thepharmaceutical compositions contain about 0.1 to about 500 μl of DNA. Insome preferred embodiments, the pharmaceutical compositions-containabout 1 to about 350 μg of DNA. In some preferred embodiments, thepharmaceutical compositions contain about 25 to about 250 μg of DNA. Insome preferred embodiments, the pharmaceutical compositions containabout 100 μg DNA.

The pharmaceutical compositions according to the present invention whichcomprise gene constructs of the invention are formulated according tothe mode of administration to be used. One having ordinary skill in theart can readily formulate a vaccine or non-immunogenic therapeutic thatcomprises a genetic construct. In cases where intramuscular injection isthe chosen mode of administration, an isotonic formulation is preferablyused. Generally, additives for isotonicity can include sodium chloride,dextrose, mannitol, sorbitol and lactose. In some cases, isotonicsolutions such as phosphate buffered saline are preferred. Stabilizersinclude gelatin and albumin. In some embodiments, a vasoconstrictionagent is added to the formulation. The pharmaceutical preparationsaccording to the present invention are provided sterile and pyrogenfree. Pharmaceutical compositions according to the invention includedelivery components in combination with nucleic acid molecules whichfurther comprise a pharmaceutically acceptable carriers or vehicles,such as, for example, saline. Any medium may be used which allows forsuccessful delivery of the nucleic acid. One skilled in the art wouldreadily comprehend the multitude of pharmaceutically acceptable mediathat may be used in the present invention. Suitable pharmaceuticalcarriers are described in Remington's Pharmaceutical Sciences, A. Osol,a standard reference text in this field, which is incorporated herein byreference.

In some embodiments, the nucleic acid molecule is delivered to the cellsin conjunction with administration of a facilitating agent. Facilitatingagents are also referred to as polynucleotide function enhancers orgenetic vaccine facilitator agents. Facilitating agents are described inU.S. Pat. No. 5,830,876 issued Nov. 3, 1998, U.S. Pat. No. 5,593,972issued Jan. 14, 1997 and International Application Serial NumberPCT/US94/00899 filed Jan. 26, 1994 (U.S. Ser. No. 08/979,385 filed Nov.29, 1997), which are each incorporated herein by reference. In addition,facilitating agents are described in U.S. Pat. No. 5,739,118 issued Apr.14, 1998, U.S. Pat. No. 5,837,533 issued Nov. 17, 1998, PCT/US95/12502filed Sep. 28, 1995 and PCT/US95/04071 filed Mar. 30, 1995, which areeach incorporated herein by reference. Facilitating agents which areadministered in conjunction with nucleic acid molecules may beadministered as a mixture with the nucleic acid molecule or administeredseparately simultaneously, before or after administration of nucleicacid molecules. In addition, other agents which may functiontransfecting agents and/or replicating agents and/or inflammatory agentsand which may be co-administered with or without a facilitating agentinclude growth factors, cytokines and lymphokines such as α-interferon,gamma-interferon, platelet derived growth factor (PDGF), GC-SF, GM-CSF,TNF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-6, IL-8, IL-10,IL-12 and B7.2 as well as fibroblast growth factor, surface activeagents such as immune-stimulating complexes (ISCOMS), Freund'sincomplete adjuvant, LPS analog including monophosphoryl Lipid A (MPL),muramyl peptides, quinone analogs and vesicles such as squalene andsqualene, and hyaluronic acid. In embodiments which relate to methods ofimmunizing, co-agents are selected which preferably enhance immuneresponses. In embodiments which relate to methods of immunosuppressing,co-agents are selected which do not enhance immune responses.

In some preferred embodiments, the genetic constructs of the inventionare formulated with or administered in conjunction with a facilitatorselected from the group consisting of benzoic acid esters, anilides,amidines, urethans and the hydrochloride salts thereof such as those ofthe family of local anesthetics.

The facilitators in some preferred embodiments may be a compound havingone of the following formulae:Ar—R¹—O—R²—R³orAr—N—R¹—R²—R³orR⁴—N—R⁵—RorR⁴—O—R¹—R⁷wherein:

Ar is benzene, p-aminobenzene, m-aminobenzene, o-aminobenzene,substituted benzene, substituted p-aminobenzene, substitutedm-aminobenzene, substituted o-aminobenzene, wherein the amino group inthe aminobenzene compounds can be amino, C₁-C₅ alkylamine, C₁-C₅, C₁-C₅dialkylamine and substitutions in substituted compounds are halogen,C₁-C₅ alkyl and C₁-C₅ alkoxy;

R¹ is C═O;

R² is C₁-C₁₀ alkyl including branched alkyls;

R³ is hydrogen, amine, C₁-C₅ alkylamine, C₁-C₅, C₁-C₅ dialkylamine;

R²+R³ can form a cyclic alkyl, a C₁-C₁₀ alkyl substituted cyclic alkyl,a cyclic aliphatic amine, a C₁-C₁₀ alkyl substituted cyclic aliphaticamine, a heterocycle, a C₁-C₁₀ alkyl substituted heterocycle including aC₁-C₁₀ alkyl N-substituted heterocycle;

R⁴ is Ar, R² or C₁-C₅ alkoxy, a cyclic alkyl, a C₁-C₁₀ alkyl substitutedcyclic alkyl, a cyclic aliphatic amine, a C₁-C₁₀ alkyl substitutedcyclic aliphatic amine, a heterocycle, a C₁-C₁₀ alkyl substitutedheterocycle and a C₁-C₁₀ alkoxy substituted heterocycle including aC₁-C₁₀ alkyl N-substituted heterocycle;

R⁵ is C═NH;

R⁶ is Ar, R² or C₁-C₅ alkoxy, a cyclic alkyl, a C₁-C₁₀ alkyl substitutedcyclic alkyl, a cyclic aliphatic amine, a C₁-C₁₀ alkyl substitutedcyclic aliphatic amine, a heterocycle, a C₁-C₁₀ alkyl substitutedheterocycle and a C₁-C₁₀ alkoxy substituted heterocycle including aC₁-C₁₀ alkyl N-substituted heterocycle; and

R⁷ is Ar, R² or C₁-C₅ alkoxy, a cyclic alkyl, a C₁-C₁₀ alkyl substitutedcyclic alkyl, a cyclic aliphatic amine, a C₁-C₁₀ alkyl substitutedcyclic aliphatic amine, a heterocycle, a C₁-C₁₀ alkyl substitutedheterocycle and a C₁-C₁₀ alkoxy substituted heterocycle including aC₁-C₁₀ alkyl N-substituted heterocycle.

Examples of esters include: benzoic acid esters such as piperocaine,meprylcaine and isobucaine; para-aminobenzoic acid esters such asprocaine, tetracaine, butethamine, propoxycaine and chloroprocaine;mera-aminobenzoic acid esters including metabuthamine and primacame; andpara-ethoxybenzoic acid esters such as parethoxycaine. Examples ofanilides include lidocaine, etidocaine, mepivacaine, bupivacaine,pyrrocaine and prilocalne. Other examples of such compounds includedibucaine, benzocaine, dyclonine, pramoxine, proparacaine, butacaine,benoxinate, carbocaine, methyl bupivacaine, butasin picrate, phenacaine,diothan, luccaine, intracaine, nupercaine, metabutoxycaine, piridocaine,biphenamine and the botanically-derived bicyclics such as cocaine,cinnamoylcocaine, truxilline and cocaethylene and all such compoundscomplexed with hydrochloride.

In preferred embodiments, the facilitator is bupivacaine. The differencebetween bupivacaine and mepivacaine is that bupivacaine has a N-butylgroup in place of an N-methyl group of mepivacaine. Compounds may haveat that N, C₁-C₁₀. Compounds may be substituted by halogen such asprocaine and chloroprocaine. The anilides are preferred.

The facilitating agent is administered prior to, simultaneously with orsubsequent to the genetic construct. The facilitating agent and thegenetic construct may be formulated in the same composition.

Bupivacaine-HCl is chemically designated as 2-piperidinecarboxamide,1-butyl-N-(2,6-dimethylphenyl)-monohydrochloride, monohydrate and iswidely available commercially for pharmaceutical uses from many sourcesincluding from Astra Pharmaceutical Products Inc. (Westboro, Mass.) andSanofi Winthrop Pharmaceuticals (New York, N.Y.), Eastman Kodak(Rochester, N.Y.). Bupivacaine is commercially formulated with andwithout methylparaben and with or without epinephrine. Any suchformulation may be used. It is commercially available for pharmaceuticaluse in concentration of 0.25%, 0.5% and 0.75% which may be used on theinvention. Alternative concentrations, particularly those between0.05%-1.0% which elicit desirable effects may be prepared if desired.According to the present invention, about 250 μg to about 10 mg ofbupivacaine is administered. In some embodiments, about 250 μg to about7.5 mg is administered. In some embodiments, about 0.05 mg to about 5.0mg is administered. In some embodiments, about 0.5 mg to about 3.0 mg isadministered. In some embodiments about 5 to 50 μg is administered. Forexample, in some embodiments about 50 μl to about 2 ml, preferably 50 μlto about 1500 μl and more preferably about 1 ml of 0.25-0.50%bupivacaine-HCl and 0.1% methylparaben in an isotonic pharmaceuticalcarrier is administered at the same site as the vaccine before,simultaneously with or after the vaccine is administered. Similarly, insome embodiments, about 50 μl to about 2 ml, preferably 50 μl to about1500 μl and more preferably about 1 ml of 0.25-0.50% bupivacaine-HCl inan isotonic pharmaceutical carrier is administered at the same site asthe vaccine before, simultaneously with or after the vaccine isadministered. Bupivacaine and any other similarly acting compounds,particularly those of the related family of local anesthetics may beadministered at concentrations which provide the desired facilitation ofuptake of genetic constructs by cells.

In some embodiments of the invention, the individual is first subject toinjection of the facilitator prior to administration of the geneticconstruct. That is, up to, for example, up to a about a week to ten daysprior to administration of the genetic construct, the individual isfirst injected with the facilitator. In some embodiments, the individualis injected with facilitator about 1 to 5 days, in some embodiments 24hours, before or after administration of the genetic construct.Alternatively, if used at all, the facilitator is administeredsimultaneously, minutes before or after administration of the geneticconstruct. Accordingly, the facilitator and the genetic construct may becombined to form a single pharmaceutical compositions.

In some embodiments, the genetic constructs are administered free offacilitating agents, that is in formulations free from facilitatingagents using administration protocols in which the genetic constructionsare not administered in conjunction with the administration offacilitating agents.

In some embodiments relating to immunization, gene constructs of theinvention may remain part of the genetic material in attenuated livemicroorganisms or recombinant microbial vectors. In addition to usingexpressible forms of CD80 CD80ΔC mutant proteins coding sequences toimprove genetic vaccines, the present invention relates to improvedattenuated live vaccines and improved vaccines which use recombinantvectors to deliver foreign genes that encode antigens. Examples ofattenuated live vaccines and those using recombinant vectors to deliverforeign antigens are described in U.S. Pat. Nos. 4,722,848; 5,017,487;5,077,044; 5,110,587; 5,112,749; 5,174,993; 5,223,424; 5,225,336;5,240,703; 5,242,829; 5,294,441; 5,294,548; 5,310,668; 5,387,744;5,389,368; 5,424,065; 5,451,499; 5,453,364; 5,462,734; 5,470,734; and5,482,713, which are each incorporated herein by reference. Geneconstructs are provided which include the nucleotide sequence thatencodes a CD80ΔC mutants protein is operably linked to regulatorysequences that can function in the vaccinee to effect expression. Thegene constructs are incorporated in the attenuated live vaccines andrecombinant vaccines to produce improved vaccines according to theinvention. Gene constructs may be part of genomes of recombinant viralvaccines where the genetic material either integrates into thechromosome of the cell or remains extrachromosomal. In some embodimentsrelating to non-immune response inducing therapy, nucleic acid moleculesthat encode CD80 C region protein may be delivered using any one of avariety of delivery components, such as recombinant viral expressionvectors or other suitable delivery means, so as to affect theirintroduction and expression in compatible host cells. In general, viralvectors may be DNA viruses such as recombinant adenoviruses andrecombinant vaccinia viruses or RNA viruses such as recombinantretroviruses. Other recombinant vectors include recombinant prokaryoteswhich can infect cells and express recombinant genes. In addition torecombinant vectors, other delivery components are also contemplatedsuch as encapsulation in liposomes, transferrin-mediated transfectionand other receptor-mediated means. The invention is intended to includesuch other forms of expression vectors and other suitable delivery meanswhich serve equivalent functions and which become known in the artsubsequently hereto. In a preferred embodiment of the present invention,DNA is delivered to competent host cells by means of an adenovirus. Oneskilled in the art would readily understand this technique of deliveringDNA to a host cell by such means. Although the invention preferablyincludes adenovirus, the invention is intended to include any viruswhich serves equivalent functions. In another preferred embodiment ofthe present invention, RNA is delivered to competent host cells by meansof a retrovirus. One skilled in the art would readily understand thistechnique of delivering RNA to a host cell by such means. Any retroviruswhich serves to express the protein encoded by the RNA is intended to beincluded in the present invention.

Some embodiments of the invention relate to proteins and methods ofusing the same. For example, in some methods of immunizing, immunogenicproteins and CD80ΔC mutant proteins are administered to individuals.Likewise, in some methods of treating autoimmune diseases and preventinggraft/transplant rejections by immunosuppression, CD80 C region proteinsare administered to individuals. As used herein, the term “proteins ofthe invention” is intended to mean immunogenic proteins, CD80ΔC mutantproteins and CD80 C region proteins which each can be produced bysimilar means and which can be formulated and administered in a similarmanner for use in methods of the invention.

Vectors including recombinant expression vectors that comprises anucleotide sequence that encodes proteins of the invention can beproduced routinely. As used herein, the term “recombinant expressionvector” is meant to refer to a plasmid, phage, viral particle or othervector which, when introduced into an appropriate host, contains thenecessary genetic elements to direct expression of a coding sequence.One having ordinary skill in the art can isolate or synthesize a nucleicacid molecule that encodes a protein of the invention and insert it intoan expression vector using standard techniques and readily availablestarting materials. The coding sequence is operably linked to thenecessary regulatory sequences. Expression vectors are well known andreadily available. Examples of expression vectors include plasmids,phages, viral vectors and other nucleic acid molecules or nucleic acidmolecule containing vehicles useful to transform host cells andfacilitate expression of coding sequences. Some embodiments of theinvention relate to recombinant expression vectors comprises anucleotide sequence that encodes a CD80ΔC mutant protein or a CD80 Cregion protein. The recombinant expression vectors of the invention areuseful for transforming hosts. The present invention relates to arecombinant expression vectors that comprises a nucleotide sequence thatencodes a CD80ΔC mutant protein, a chimeric protein which comprises aCD80ΔC mutant protein, or a CD80 C region protein.

The present invention relates to a host cell that comprises therecombinant expression vector that includes a nucleotide sequence thatencodes a CD80ΔC mutant protein, a chimeric protein which comprises aCD80ΔC mutant protein or a CD80 C region protein. Host cells for use inwell known recombinant expression systems for production of proteins arewell known and readily available. Examples of host cells includebacteria cells such as E. coli, yeast cells such as S. cerevisiae,insect cells such as S. frugiperda, non-human mammalian tissue culturecells chinese hamster ovary (CHO) cells and human tissue culture cellssuch as HeLa cells.

In some embodiments, for example, one having ordinary skill in the artcan, using well known techniques, insert DNA molecules into acommercially available expression vector for use in well knownexpression systems. For example, the commercially available plasmidpSE420 (Invitrogen, San Diego, Calif.) may be used for production of aCD80ΔC mutant protein in E. coli. The commercially available plasmidpYES2 (Invitrogen, San Diego, Calif.) may, for example, be used forproduction in S. cerevisiae strains of yeast. The commercially availableMAXBAC™ complete baculovirus expression system (Invitrogen, San Diego,Calif.) may, for example, be used for production in insect cells. Thecommercially available plasmid pcDNA I or pcDNA3 (Invitrogen, San Diego,Calif.) may, for example, be used for production in mammalian cells suchas Chinese Hamster Ovary cells. One having ordinary skill in the art canuse these commercial expression vectors and systems or others to produceproteins of the invention using routine techniques and readily availablestarting materials. (See e.g., Sambrook et al., Molecular Cloning aLaboratory Manual, Second Ed. Cold Spring Harbor Press (1989) which isincorporated herein by reference.) Thus, the desired proteins can beprepared in both prokaryotic and eukaryotic systems, resulting in aspectrum of processed forms of the protein.

One having ordinary skill in the art may use other commerciallyavailable expression vectors and systems or produce vectors using wellknown methods and readily available starting materials. Expressionsystems containing the requisite control sequences, such as promotersand polyadenylation signals, and preferably enhancers, are readilyavailable and known in the art for a variety of hosts. See e.g.,Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. ColdSpring Harbor Press (1989).

The expression vector including the DNA that encodes a protein of theinvention is used to transform the compatible host which is thencultured and maintained under conditions wherein expression of theforeign DNA takes place. The protein of the invention thus produced isrecovered from the culture, either by lysing the cells or from theculture medium as appropriate and known to those in the art. One havingordinary skill in the art can, using well known techniques, isolate theprotein of the invention that is produced using such expression systems.The methods of purifying proteins of the invention from natural sourcesusing antibodies which specifically bind to such protein are routine asis the methods of generating such antibodies (See: Harlow, E. and Lane,E., Antibodies: A Laboratory Manual, 1988, Cold Spring Harbor LaboratoryPress which is incorporated herein by reference.). Such antibodies maybe used to purifying proteins produced by recombinant DNA methodology ornatural sources.

Examples of genetic constructs include coding sequences which encode aprotein of the invention and which are operably linked to a promoterthat is functional in the cell line into which the constructs aretransfected. Examples of constitutive promoters include promoters fromcytomegalovirus or SV40. Examples of inducible promoters include mousemammary leukemia virus or metallothionein promoters. Those havingordinary skill in the art can readily produce genetic constructs usefulfor transfecting with cells with DNA that encodes proteins of theinvention from readily available starting materials. Such geneconstructs are useful for the production of proteins of the invention.

In addition to producing proteins of the invention by recombinanttechniques, automated peptide synthesizers may also be employed toproduce proteins of the invention. Such techniques are well known tothose having ordinary skill in the art and are useful if derivativeswhich have substitutions not provided for in DNA-encoded proteinproduction.

The proteins of the invention may be prepared by any of the followingknown techniques. Conveniently, the proteins of the invention may beprepared using the solid-phase synthetic technique initially describedby Merrifield, in J. Am. Chem. Soc., 15: 2149-2154 (1963) which isincorporated herein by reference. Other protein synthesis techniques maybe found, for example, in M. Bodanszky et al., (1976) Peptide Synthesis,John Wiley & Sons, 2d Ed. which is incorporated herein by reference;Kent and Clark-Lewis in Synthetic Peptides in Biology and Medicine, p.295-358, eds. Alitalo, K., et al. Science Publishers, (Amsterdam, 1985)which is incorporated herein by reference; as well as other referenceworks known to those skilled in the art. A summary of synthesistechniques may be found in J. Stuart and J. D. Young, Solid PhasePeptide Synthelia, Pierce Chemical Company, Rockford, Ill. (1984) whichis incorporated herein by reference. Synthesis by solution methods mayalso be used, as described in The Proteins, Vol. II, 3d Ed., p. 105-237,Neurath, H. et al., Eds., Academic Press, New York, N.Y. (1976) which isincorporated herein by reference. Appropriate protective groups for usein such syntheses will be found in the above texts, as well as in J. F.W. McOmie, Protective Groups in Organic Chemistry, Plenum Press, NewYork, N.Y. (1973) which is incorporated herein by reference.

In general, these synthetic methods involve the sequential addition ofone or more amino acid residues or suitable protected amino acidresidues to a growing peptide chain. Normally, either the amino orcarboxyl group of the first amino acid residue is protected by asuitable, selectively-removable protecting group. A different,selectively removable protecting group is utilized for amino acidscontaining a reactive side group, such as lysine.

Using a solid phase synthesis as an example, the protected orderivatized amino acid is attached to an inert solid support through itsunprotected carboxyl or amino group. The protecting group of the aminoor carboxyl group is then selectively removed and the next amino acid inthe sequence having the complementary (amino or carboxyl) group suitablyprotected is admixed and reacted with the residue already attached tothe solid support. The protecting group of the amino or carboxyl groupis then removed from this newly added amino acid residue, and the nextamino acid (suitably protected) is then added, and so forth. After allthe desired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently, to provide the final peptide.The peptide of the invention are preferably devoid of benzylated ormethylbenzylated amino acids. Such protecting group moieties may be usedin the course of synthesis, but they are removed before the peptides areused. Additional reactions may be necessary, as described elsewhere, toform intramolecular linkages to restrain conformation.

In some embodiments, proteins may be produced in transgenic animals. Thepresent invention relates to a transgenic non-human mammal thatcomprises the recombinant expression vector that comprises a nucleicacid sequence that encodes a CD80ΔC mutant protein or CD80 C regionprotein. Transgenic non-human mammals useful to produce recombinantproteins are well known as are the expression vectors necessary and thetechniques for generating transgenic animals. Generally, the transgenicanimal comprises a recombinant expression vector in which the nucleotidesequence that encodes the CD80ΔC mutant protein or CD80 C region proteinis operably linked to a mammary cell specific promoter whereby thecoding sequence is only expressed in mammary cells and the recombinantprotein so expressed is recovered from the animal's milk. One havingordinary skill in the art using standard techniques, such as thosetaught in U.S. Pat. No. 4,873,191 issued Oct. 10, 1989 to Wagner andU.S. Pat. No. 4,736,866 issued Apr. 12, 1988 to Leder, both of which areincorporated herein by reference, can produce transgenic animals whichproduce a CD80ΔC mutant protein or CD80 C region protein. Preferredanimals are goats, and rodents, particularly rats and mice.

Conservative substitutions of amino acid sequences of proteins of theinvention are contemplated. As used herein, the term “conservativesubstitutions” is meant to refer to amino acid substitutions of CD80residues with otherresidues which share similar structural and/or chargefeatures. Those having ordinary skill in the art can readily designproteins of the invention with conservative substitutions for aminoacids based upon well known conservative groups.

The pharmaceutical compositions of the present invention may beadministered by any means that enables the active agent to reach theagent's site of action in the body of a mammal. The pharmaceuticalcompositions of the present invention may be administered in a number ofways depending upon whether local or systemic treatment is desired andupon the area to be treated. Administration may be topical (includingophthalmic, vaginal, rectal, intranasal, transdermal), oral orparenteral. Because peptides are subject to being digested whenadministered orally, oral formulations are formulated to entericallycoat the active agent or otherwise protect it from degradation in thestomach (such as prenuetralization). Parenteral administration includesintravenous drip, subcutaneous, intraperitoneal or intramuscularinjection, pulmonary administration, e.g., by inhalation orinsufflation, or intrathecal or intraventricular administration. Inpreferred embodiments, parenteral administration, i.e., intravenous,subcutaneous, transdermal, intramuscular, is ordinarily used to optimizeabsorption. Intravenous administration may be accomplished with the aidof an infusion pump. The pharmaceutical compositions of the presentinvention may be formulated as an emulsion.

One skilled in the art would readily comprehend the multitude ofpharmaceutically acceptable media that may be used in the presentinvention. Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, A. Osol, a standard reference text in thisfield, which is incorporated herein by reference. Formulations fortopical administration may include transdermal patches, ointments,lotions, creams, gels, drops, suppositories, sprays, liquids andpowders. Conventional pharmaceutical carriers, aqueous, powder or oilybases, thickeners and the like may be necessary or desirable.Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachcts or tablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Compositions forparenteral, intravenous, intrathecal or intraventricular administrationmay include sterile aqueous solutions which may also contain buffers,diluents and other suitable additives and are preferably sterile andpyrogen free. Pharmaceutical compositions which are suitable forintravenous administration according to the invention are sterile andpyrogen free. For parenteral administration, the peptides of theinvention can be, for example, formulated as a solution, suspension,emulsion or lyophilized powder in association with a pharmaceuticallyacceptable parenteral vehicle. Examples of such vehicles are water,saline, Ringer's solution, dextrose solution, and 5% human serumalbumin. Liposomes and nonaqueous vehicles such as fixed oils may alsobe used. The vehicle or lyophilized powder may contain additives thatmaintain isotonicity (e.g., sodium chloride, mannitol) and chemicalstability (e.g., buffers and preservatives). The formulation issterilized by commonly used techniques. For example, a parenteralcomposition suitable for administration by injection is prepared bydissolving 1.5% by weight of active ingredient in 0.9% sodium chloridesolution

The pharmaceutical compositions according to the present invention maybe administered as a single dose or in multiple doses. Thepharmaceutical compositions of the present invention may be administeredeither as individual therapeutic agents or in combination with othertherapeutic agents. The treatments of the present invention may becombined with conventional therapies, which may be administeredsequentially or simultaneously.

Dosage varies depending upon known factors such as the pharmacodynamiccharacteristics of the particular agent, and its mode and route ofadministration; age, health, and weight of the recipient; nature andextent of symptoms, kind of concurrent treatment, frequency oftreatment, and the effect desired. Formulation of therapeuticcompositions and their subsequent administration is believed to bewithin the skill of those in the art. Usually, the dosage of peptide canbe about 1 to 3000 milligrams per 50 kilograms of body weight;preferably 10 to 1000 milligrams per 50 kilograms of body weight; morepreferably 25 to 800 milligrams per 50 kilograms of body weight.Ordinarily 8 to 800 milligrams are administered to an individual per dayin divided doses 1 to 6 times a day or in sustained release form iseffective to obtain desired results.

Depending upon the method for which the protein or proteins are beingadministered, the pharmaceutical compositions of the present inventionmay be formulated and administered to most effectively. Modes ofadministration will be apparent to one skilled in the art in view of thepresent disclosure.

The methods of the present invention are useful in the fields of bothhuman and veterinary medicine. Accordingly, the present inventionrelates to genetic immunization of mammals, birds and fish. The methodsof the present invention can be particularly useful for mammalianspecies including human, bovine, ovine, porcine, equine, canine andfeline species.

The Examples set out below include representative examples of aspects ofthe present invention. The Examples are not meant to limit the scope ofthe invention but rather serve exemplary purposes. In addition, variousaspects of the invention can be summarized by the following description.However, this description is not meant to limit the scope of theinvention but rather to highlight various aspects of the invention. Onehaving ordinary skill in the art can readily appreciate additionalaspects and embodiments of the invention.

EXAMPLE

In an effort to determine why CD86, but not CD80, is required foraugmentation of T-cell responses, and to further study the structurefunction analysis of CD80 and CD86 which has revealed several criticalareas involved in binding to CD28 and CTLA-4 including residues found onboth the V- and C-domains of CD80, the role of different regions of CD80and CD86 molecules in T-cell activation were examined usingcoimmunization of mice with DNA immunogen and DNA encoding chimeric ortruncated forms of CD80 and CD86 molecules.

Methods

Preparation of constructs:

A DNA vaccine construct encoding for the HIV-1_(MN) envelope protein(pcEnv) was prepared as described in U.S. Pat. No. 5,593,972. Human CD80and CD86 genes were cloned from B cell cDNA library (Clontech, PaloAlto, Calif.) and placed into pSRαneo1+, an expression vector. Morespecifically, both CD80 and CD86 genes were PCR amplified as describedin Kim, et al. (1997) Nature Biot. 15: 641-645, which is incorporatedherein by reference, and ligated into pSRαneo1+ downstream of the SRαpromoter to make pCD80 and pCD86 expression vectors. The chimeric andtruncated variants of these two genes were generated by PCRamplification using the Expand™ High Fidelity Polymerase system(Boehringer-Mannheim, Germany). For construction of all these forms ofcostimulatory molecules, pCD80 or pCD86 were used as the PCR templates.The following primers have been used in these reactions:

A: CTGCTTGCTCAACTCTACGTC—SEQ ID NO:1 (forward, vector)

B: CTGAAGTTAGCTTTGACTGATAACG—SEQ ID NO:2 (reverse, CD80)

C: GCAATAGCATCACAAATTTCA—SEQ ID NO:3 (reverse, vector)

D: CAGTCAAAGCTAACTTCAGTCAACC—SEQ ID NO:4 (forward, CD86)

E: GGGAAGTCAGCAAGCACTGACAGTTC—SEQ ID NO:5 (reverse, CD86)

F: TCAGTGCTTGCTGACTTCCCTACACC—SEQ ID NO:6 (forward, CD80)

G: TCTTGCTTGGCTTTGACTGATAACGTCAC—SEQ ID NO:7 (reverse, CD80)

H: TCAGTCAAAGCCAAGCAAGAGCATTTTCC—SEQ ID NO:8 (forward, CD80)

I: TCCTCAAGCTCAAGCACTGACAGTTC—SEQ ID NO:9 (reverse, CD86)

J: TCAGTGCTTGAGCTTGAGGACCC—SEQ ID NO: 10 (forward, CD86)

K: TCTGGATCCTCATCTTGGGGCA—SEQ ID NO: 11 (reverse, CD80)

L: TCTGGATCCTCATTTCCATAG—SEQ ID NO:12 (reverse, CD86)

The V-domain of CD80 was amplified using A and B primers, the C-domain,transmembrane (TM) and cytoplasmic tail (T) of CD86 were amplified usingC and D primers. These fragments were then purified, combined and usedas templates in second step PCR reaction using forward(CTGCTTGCTCAACTCTACGTC—SEQ ID NO:1) and reverse(GCAATAGCATCACAAATTTCA—SEQ ID NO:3) primers. The PCRproduct was ligatedinto the pSRaneol+vector and the resultant plasmid (pV80C86T86) encodesa chimeric costimulatory molecule expressing the V-domain of CD80 andC—, TM-, and T-regions of CD86. The chimeric crossover point is at theconserved alanine 106 in CD80 and alanine 111 in CD86 position andrespects the exon boundary.

The next plasmid pV86CSOT80 which encoded CD86 V-domain and CD80 C—,TM-, and T-regions was constructed by amplification of CD86 V-region,using A and E primers. The PCR fragment encoding C. TM-, and T-domainsof CD80 was amplified using C and F primers. The second stage PCR andcloning was performed as mentioned above.

Truncated forms of costimulatory molecules without C-domain (pV80CAT80,pV86CAT86) were also prepared by two step PCR technique. In case ofpV80CAT80 the V-domain was amplified using the A and G primers, whereasin case of pV86C(T86 V-domain was amplified using A and I primers. TheTM/T fragments of both C-domain truncated molecules were amplified usingC/H primers in case of pV80C(T80 and C/J in case of pV86CDT86. Theresultant constructs were prepared by amplification and cloning the PCRproducts into the pSRaneol+ expression vector. All constructs wereverified by sequence to be faithful to the original wildtype CD80 andCD86 templates. The resulting deletion mutants lacked amino acidaspartatel 07 through threonine 200 in CD80 and alanine 111 throughisoleucine 211 in CD86. Of note both molecules were constructed toretain 6 to 7 membrane proximal amino acids of the respective C-domain.

Finally, the T-region deletion of pCD80 (pV80C80T( ) and pCD86(pV86C86TΔ) were generated by a single step PCR using in both cases A asforward and K and L as reverse primers, respectively. The PCR productswere cloned into the pSR(neo1+ vector. The encoded CD80 proteinterminates after the first cytoplasmic tail amino acid residue,arginine. The resulting gene for CD86 molecule terminated afternucleotide 942 preserving the first lysine in the cytoplasmic tail.

All chimeric and truncated constructs as well as the wildtype moleculeswere cloned into the SRaneol+vector. Gene expression is under thecontrol of the SRα promoter which is composed of the simian virus 40(SV40) early promoter and the R-segment and part of the U5 sequence(R-U5′) of the long terminal repeat of human T-cell leukemia virustype 1. All constructs were verified by sequencing to be faithful to theoriginal wildtype CD80 and CD86 templates.

Expression of Plasmids:

Expression of these constructs were analyzed by immunofluorescence andflow cytometry (FACS) assays, using human rhabdomyosarcoma (RD) cellstransfected with experimental or control plasmids. Cells weretransfected by electroporation using 500 μF capacitance and 0.25 voltageusing Gene Pulse (Bio-Rad, Hercules, Calif.).

For immunofluorescence assay transfected cells were incubated for 2 daysand than transferred into Falcon® culture slides (Becton Dickinson,Bedford, Mass.). The following day the cells were washed, fixed withmethanol (30′, RT), and incubated with anti-CD80 (Coulter, Miami, Fla.)or CD86 (Pharmingen, San Diego, Calif.) monoclonal antibodies (1.5 hrs,37° C.). The slides were washed and stained with goat-anti mouse IgG(Boehringer Mannheim, Indianapolis, Ind.) during 1.5 hrs at 37° C. Theslides were viewed with a Nikon OPTIPHOT fluorescence microscope (NikonInc., Tokyo, JAPAN) and photographs were obtained.

For FACS analysis RD cells were transfected with mixture of constructsencoding CD80 or CD86 molecules (2 μg) and green fluorescent proteinexpression vector {10 μg (pcGFP from Clontech, Palo Alto, Calif.)}. Thelatter was used as a control plasmid for calculation of the efficacy oftransfection. The expression of experimental plasmids was confirmedusing monoclonal antibodies to the V-domain of CD80 or CD86 molecules(both from Pharmingen, San Diego, Calif.) conjugated with PE. Briefly, 1μg of either anti-B7 antibodies were added to transfected or controlcells (10×10⁵). Data were analyzed by FACScan with CELLQueSt™ dataacquisition and software (Becton Dickinson Immunocytometry Systems, SanJose, Calif.). The efficacy of transfection (fluorescence intensity) ofcells expressing different B7 molecules was measured in the populationof cells expressing GFP.

Immunization of Animals:

Each Balb/c mouse received three intramuscular injections (two weeksapart) with 50 μg of each DNA construct resuspended in 100 μl ofphosphate buffered saline (PBS) and 0.25% bupivacaine-HCl (Sigma, St.Louis, Mo.). This dose was selected to maximize the enhancement ofanti-viral immune responses provided by the simultaneous delivery ofdifferent molecular adjuvants (i.e. mutated, truncated, or wildtypecostimulatory genes). 50 μg of plasmid, encoding HIV-1 gp160 envelope(pcEnv) was injected alone or as a mixture of pcEnv and 50 μg of thevarious CD80/86 constructs (molecular adjuvants). As a control naivemice and animals injected with control vectors were used. In severalexperiments animals were injected three times with the mixture of twomolecular adjuvants (100 μg total) plus pcEnv (50 μg). Two weeks afterthe last injection, splenocytes from all experimental and controlanimals were isolated and used for detection of T-cell responses andcytokine production.

Immunohistochemical Assays on Muscle Cells:

Immunized leg muscle was examined immunohistochemically for detection ofinfiltration (presence of lymphocytes in muscle). Briefly, mousequadriceps muscle was inoculated with 50 μg of pcEnv mixed with 50 μg ofexperimental or control plasmids. Seven days following inoculation, themice were sacrificed and the quadriceps muscles were removed. The freshmuscle tissue was then frozen in O.C.T. compound (Sakura Finetek USA,Inc., Torrance, Calif.) and four micron frozen sections were made. Thedegree of inflammation was determined by examining hematoxylin and eosin(H&E) stained muscle sections.

Cytotoxic T Lymphocyte Assay:

A five hour ⁵¹Cr release CTL assay was performed. Briefly, the effectorswere stimulated six days in the presence of stimulator cells and 10%RAT-T-STIM without Con A (Becton Dickinson Labware, Bedford, Mass.). Forantigenic stimulation, 0.1% glutaraldehyde fixed P-815 cells infectedwith recombinant vaccinia virus which express HIV-1 envelope protein(vMN462) (NIH AIDS Research and Reference Reagent Program) were used. Astarget cells, P-815 cells infected with the recombinant (vMN462,specific) or with wildtype (WR, non-specific) vaccinia virus were used.Both target cells were labeled with 100 (Ci/ml Na₂51 CrO₄ and mixed witheffector cells at effector: target (E:T) ratios ranging from 100:1 to12.5:1. The percent specific lysis was determined as described in Kim,1997 supra. Maximum and minimum release was determined by lysis oftarget cells in 10% Triton X-100 and medium, respectively. An assay wasnot considered valid if the value for the ‘spontaneous release’ countswere in excess of 20% of the ‘maximum release’. To calculate specificlysis of targets, the percent lysis of non-specific targets wassubtracted from the percent lysis of specific targets. In someexperiments CD8+ T-cells were removed from culture of splenocytes bytreatment with anti-CD8 monoclonal antibodies (53-6.7, ATCC) followed byincubation with non-toxic rabbit complement (Sigma).

Cytokine Production:

The level of various cytokines released by immune cells reflects thedirection and magnitude of the immune response. Therefore, supernatantfrom the effector cells stimulated in vitro for CTL assay was collectedand tested them for the release of yIFN, IL-4, and IL-12 using availableELISA kits (Biosource, Camarillo, Calif.).

Results

Expression of Plasmids, Encoding Wildtypes and Mutated Forms of CD80 andCD86:

The expression of different forms of CD80 or CD86 constructs into the RDmuscle tumor cells transiently transfected with control or experimentalplasmids was analyzed first. Using an immunofluorescence technique, iswas observed that experimental, but not control cells (transfected withvector alone) produced wildtype as well as all the different forms ofmutated costimulatory molecules. The transfection efficiencies of thesemolecules were studied by FACS analysis. In these experiments a mixtureof control plasmid, encoding GFP and experimental constructs, encodingdifferent forms of costimulatory molecules, were transfected. Thefluorescence intensity of cells expressing B7 molecules was detectedonly in the population of cells expressing GFP. The results demonstratedthat majority of chimeric and truncated forms of CD86 and CD80 and CD86wildtype molecules have been expressed similarly (Table 3). Only twoconstructs, encoding CD80 wildtype (pCD80) and cytoplasmic tail deletedCD86 (pV86C86TA) were expressed on the surface of transfected cellsrelatively higher than other plasmids.

Both CD86 and CD80 V-Domains are Important for the Activation ofVirus-Specific CTL Response and Th1 Cytokine Production

The B7 molecules play a critical role in inducing antigen-specificT-cell activation via triggering of appropriate ligands expressed onthese cells. Earlier, it was reported that co-administration of wildtypeCD86, but not CD80 cDNA along with DNA immunogen enhancedantigen-specific T-cell responses (Kim et al. 1997 Supra). To determinethe role of the V-region of CD86 in this activation, these domains ofCD80 and CD86 were exchanged (Table 3) and coimmunized mice withconstructs encoding these molecules along with plasmid encoding viralproteins. As a positive control, constructs expressing wildtype CD80 andCD86 were coinjected along with DNA immunogen, negative control micereceived only the vector. Two weeks after the last immunizationanti-virus CTL responses were analyzed in the cultures of splenocytes.

A background level of specific killing was observed in splenocytesobtained from the control animals and a low level killing was observedin animals coimmunized with pcEnv or pcEnv plus pCD80. However, micecoimmunized with pcEnv and pCD86 resulted in a high level ofenvelope-specific CTL (Table 4). Therefore, using the CD80 and CD86genes inserted in the pSRaneol+ vector, instead of the previously usedpcDNA3 vector, CD80 and CD86 were confirmed to play differential rolesin the modulation of cellular immune responses following DNAvaccination. Anti-viral CTL responses were next analyzed in miceimmunized with chimeric molecules. Communization of mice with pV86C80T80and pcEnv did not generate virus-specific cytotoxic cells, butimmunization of mice with mixture of pV80C86T86 and pcEnv induced morethan 40% anti-HIV-1 CTL activity at E:T ratio 1:100. This response wassimilar to anti-viral CTL activity in mice coinjected with pcEnv pluspCD86 (Table 4). Thus, V region of CD80 was as important forantigen-specific T-cell activation as the V-region of the CD86 molecule,if it was expressed with the C-domain and cytoplasmic tail of the CD86molecule. However, the V-domain of CD86 was functionally silent whenexpressed with C-domain and cytoplasmic tail of CD80. These results weresupported by cytokine production data. Supernatant from splenocytesobtained from mice injected with pcEnv and pcEnv plus pCD80 induced lowlevel of IL12, but not γIFN or IL4 production (Table 4). In contrast,coimmunization with pCEnv+pCD86 and pcEnv+pV80C86T86 induced significantantigen-specific enhancement of both γIFN and IL12 (Table 4), but notIL4 production.

These results would suggest that the C-domain and/or cytoplasmic tail ofCD86 are important in positive signaling to T-cells, whereas the samedomains of CD80 are not. Alternatively, the C-domain and/or cytoplasmictail of CD80 might be involved in providing of negative signals toT-cells.

Cytoplasmic Tail of CD86 Crucial for Antigen-Specific T-Cell Activation

It has been demonstrated that the cytoplasmic tail of B7 is required forin vitro T-cell costimulation by allowing for ligand clustering on thecell surface. Thus, to demonstrate the role of the cytoplasmic tails ofthe B7 in T-cell costimulation, cytoplasmic tail deleted mutants of B7were constructed and coinjected mice with these plasmids (pV80C80TAorpV86C86TA) and pcEnv. Both constructs, encode truncated forms of CD80or CD86 molecules induced lower level killing, whereas animalscoimmunized with a mixture of pcEnv and pCD86 demonstrated stronganti-viral CTL responses (Table 4). Supportive data was generated byanalyzing Th1 cytokine production.

Both CD80 and CD86 constructs without cytoplasmic tails did not enhanceγIFN production after coinjection with DNA vaccine. Communization ofmice with pV86C86TA induced a small increase of IL12 production comparedwith mice injected with pcEnv or pcEnv plus pV80C80TΔ: Importantly,control animals coimmunized with pcEnv plus DNA encoding wildtype ofCD86 induce significant enhancement of both γIFN and IL12 cytokineproduction (Table 4). Thus, the cytoplasmic tail of CD86 molecule wasimportant for T-cell activation. However, mice coimmunized with pcEnvplus pV80C80TΔ did not induce T-cell activation. Therefore, theinvolvement of the C-domain and/or cytoplasmic tail of CD80 in negativesignaling remained undetermined. To resolve this question next C-domaindeletion mutants of CD80 and CD86 were constructed.

C-Domain, but not Cytoplasmic Tail of CD80, is Involved in Providing ofa Negative Signal to T-Cells.

In the next set of experiments mice were coimmunized with pcEnvimmunogen and plasmids, encoding only the V-domain and cytoplasmic tailof either CD80 (pV80CΔT80) or CD86 (pV86CΔT86) molecules. As controls,mice injected with only pcEnv or with pcEnv plus either pCD80 or pCD86.The antigen specific anti-viral CTL responses from these experiments arepresented in FIG. 1. The adjuvant effect of CD86 was dramatic and wasretained in lesser extend in the CD86 C-domain deletion molecule. Insharp contrast to the very poor effect in T-cell activation induced bywildtype and cytoplasmic tail deleted forms of CD80 (FIG. 1, Table 4),pV80CAT80 was very effective at costimulating the anti-Env CTL response(FIG. 1), demonstrating gain of function through the loss of C-domain.To further investigate the enhancement of cellular immunity, productionof Th1 cytokines was investigate using splenocytes of mice immunizedwith pcEnv and plasmids, encoding C-domain deleted CD80 or CD86molecules. As controls, mice coimmunized with pcEnv plus either pCD80 orpCD86. Both pV86CΔT86 and pV80CΔT80 chimeric genes as well as DNAencoded wildtype CD86 coinjected along with pcEnv induced Th1 lymphokineproduction equally well (FIGS. 2A and 2B).

These results demonstrate that the cytoplasmic tail of CD80 isfunctional and is important for T-cell activation in vivo. Moreimportantly, the data support the conclusion that the C-domain of CD80,but not CD86, can provide a “negative” signal to T-cells. Next, theinhibitory role of the CD80 C-domain in T-cell activation was analyzed.

C-Domain of CD80 Inhibits T-Cell Activation by CD86 Molecules.

To demonstrate the involvement of the C-domain of CD80 in providing anegative signal to antigen-specific T-cells, animals were immunized withthe DNA immunogen and a combination of molecular adjuvants. Experimentalmice were coimmunized with DNA immunogen and mixture of pCD86 with pCD80or pCD86 with pV80CDT80. Control animals were injected with only pcEnvor coinjected with pcEnv plus pCD86, pCD80 or pV80C(T80. Anti-viral CTLassays were performed with splenocytes obtained from experimental andcontrol mice.

As observed before coimmunization of mice with DNA immunogen and pCD86or pV80C(T80 induced significant enhancement of CTL activity (FIG. 3).However, mice coimmunized with pcEnv and combination ofwildtypesmolecular adjuvants (pCD86+pCD80) did not enhance the CTL response(lysis was not more than in control group with pcEnv). Importantly, thecombination of pCD86 and pV80CDT80 still induced an adjuvant effect invaccinated animals and this effect was similar to effect induced inanimals immunized with pCD86 plus DNA immunogen. Therefore, wildtypeCD80, but not C-domain deleted mutant CD80 inhibited enhancement ofanti-viral CTL responses when codelivered with the DNA immunogen. Toverify the role of CD8′ T cells in the cytotoxic activity observed, CTLactivity was measured after the removal of this population of cells. Thesplenocytes were treated with anti-CD8 monoclonal antibodies andnon-toxic rabbit complement. The removal of CD8⁺ T-cells resulted in thesuppression of the anti-viral CTL activity in mice coinjected with DNAimmunogen and pCD86. Again no anti-HIV-1 CTL activity was observed inmice coimmunized with pcEnv+pCD80+pCD86 (FIG. 4).

Expression of C-Domain Deleted CD80 Induced Greater Infiltration ofLymphocytes into the Muscle of Immunized Animals Than Expression ofWildtype CD80.

A markedly greater infiltration of lymphocytes into the muscle of miceimmunized with pCEnv+pCD86 than in control or pCEnv+pCD80 immunizedanimals has been reported. The infiltrating cells included both CD4⁻ andCD8⁺T cells. Thus, to further determine the ability of C-domain deletedCD80 molecules to interact with T cells, infiltration of cells into themuscle tissue after coinjection of mice with pcEnv plus pV80CAT80 wasinvestigate. As a control, animals injected with vector alone or amixture of pcEnv plus pCD80 or pCD86 were used. Coinjection of mice withpCEnv+pCD86, but not pcEnv+pCD80 induced a dramatic infiltration oflymphocytes into the muscle tissue (FIG. 5). Normal animals developedvirtually no infiltration. More importantly, infiltration was muchgreater in the muscle of mice coimmunized with DNA immunogen andpV80CΔT80. Therefore, the deletion of the C-domain changed thecostimulatory properties of CD80 to resemble those of a wildtype ofCD86.

CD80 and CD80 C-Domain Deleted Mutants Have Different Binding Affinityto CTLA-4

As demonstrated above, communization of mice with DNA immunogen andpCD86 or pV80CAT80 enhanced CTL activity, Th1 cytokine production, andinfiltration of cells in the site of injection. In contrast,immunization with DNA immunogen and pCD80 did not have a similar effect.We postulate that the deletion of the C-domain of CD80 results in amolecule with decreased affinity for CTLA-4. This results in the abilityto provide a potent negative signal to T cells. We used surface plasmonresonance to compare differences in CTLA-4 binding affinity between CD80and the CD80 C-domain deletion mutant. Typically receptor(counterreceptor) molecules are immobilized to the sensor surface anddifferent concentration of the counterreceptor (receptor) continuouslyflow through this sensor. Using this methodology it was shown that humansoluble CTLA-4 bound to soluble CD80 with solution K_(D) of 0.2-0.4 μM.Cells expressing the ligand of interest were immobilized on the surfaceof the. Cell immobilization allows maximum access of the cell surfaceCD-80 epitopes to the bulk of soluble CTLA-4-Ig with minimalconformational distortion of ligands. Lateral diffusion within themembrane bilayer allows for physiologic oligomerization of the CD80ligands upon binding to CTLA-4. Using this approach, CTLA-4-Ig wasdemonstrated to specifically binds to RD cells, transfected with boththe CD80 wildtype and CD80 C-domain deletion molecules. Importantly thisbinding was concentration dependent. After subtraction of thenon-specific signal from the binding of the monoclonal antibodiesaffinities were calculated. Association of CTLA-4-Ig with the wildtypeCD80 receptor is 5 times faster than with the mutant CD80 (k_(on)parameter), while dissociation of CTLA-4/CD80 complex is 2.8 timesslower for the wildtype receptor (k_(off) parameter). Because of thesedifferences in kinetics, the CTLA-4-Ig/CD80 wildtype interaction is 14times stronger than with CD80 mutant as reflected in K_(D). Bydefinition k_(off) and k_(on) parameters measured by Biacore do notdepend on the number of receptors, expressed at the cell surface. Thus,deletion of the constant region of CD80 receptor has a profound effecton the CD80-CTLA-4 interaction itself.

Discussion

Current evidence indicates that one of the most important costimulatorypathways for T cell activation involves CD80 and CD86 expression on APC.These molecules interact with T cell surface receptors (CD28 or CTLA-4)and provide secondary signals important for proliferation and cytokinesecretion. The critical costimulatory signal for T cell activation isprovided through CD28 after binding to the B7 ligands. In contrast,CTLA-4 primarily induces an inhibitory signal. The evidence defining thefunctional roles of the CD80 and CD86 molecules is more complicated. Itwas demonstrated in several mainly in vitro models that both CD80 andCD86 have critical roles in the activation of T cells. However,expression of CD86 earlier than CD80 strongly suggesting that CD86 playsa more significant role in initiating T-cell immune responses.Differences between CD80 and CD86 can be inferred by data on the bindingkinetics of costimulatory molecules with CD28 and CTLA-4. Recently,functional differences between CD80 and CD86 molecules have also beendocumented in many model systems including DNA immunization.

The CD86 molecule, but not CD80, is more important in providing ofsecondary signals to T cells following DNA vaccination of mice. However,CD80 as well as CD86 has been reported to enhance CTL responses when itwas coexpressed with plasmid DNA encoding a minigene. This result isdifferent than other reported results suggesting that free epitopesrather than natural antigens can behave uniquely. The similarity of theresults from different groups are instructive, suggesting thatexpression of CD86 might be more important than CD80 in initiating andexpanding in vivo cellular immune responses.

To further investigate the role of CD80 and CD86 molecules in T-cellactivation, several chimeric and deletional mutant forms of CD80 andCD86 (Table 3) were constructed. These plasmids were coinjected in micewith an HIV-1/Env DNA immunogen and CTL activity as well as Th1lymphokine production were determined. Chimeric pV80C86T86 as well aspCD86, but not pCD80 support the activation of T-cells (Table 4). Thisis significant since both the CD80 and CD86 V-domains can activateT-cells. These results also suggested that the C-domain and thecytoplasmic tail of CD86 can support T-cell activation. Of note, theopposite chimeric construction (pV86C80T80) did not enable the necessarycostimulatory signal for antigen-specific T-cell stimulation. Thisraised the possibility that the C-domain and/or cytoplasmic tail of CD80may play an important inhibitory role in T cell activation. Deletionalmutants of the B7 molecules which lacked the cytoplasmic tail orC-domains were then used. This allowed the direct assessment of the roleof these molecular regions in T-cell activation or inhibition.

It was observed in vivo that the cytoplasmic tail of CD86 is absolutelynecessary for anti-viral CTL responses and for Th1 cytokine production(Table 4). However, failure of the CD80 cytoplasmic tail deletionmolecule (pV80C80TA) to induce T-cell activation did not allow themapping of the inhibitory region of this molecule to the CD80 C-domainor cytoplasmic tail. This question was resolved by coimmunization ofmice with the genes encoding the immunogen and the C-domain deleted CD80molecule (FIGS. 1, 2A and 2B). A dramatic enhancement of T-cellactivation in mice coinjected with pV80CAT80 and pCEnv was observed. Thecytoplasmic tail of CD80, like the cytoplasmic tail of CD86, appears tobe involved in T cell activation and is not involved in the inhibitionof T-cell activation. The cytoplasmic tail played an important role inboth redistribution and oligomerization of this molecule on the surfaceof professional APC. Importantly it has been demonstrated also thatactivation of CD80 transfected cells with ionomycin and or PMA resultedin the association of a 30 kDa phosphoprotein with the CD80 cytoplasmictail. Similar size of protein that was inducibly phosphorylated ontyrosine after CD80 cross-linking has been reported. Finally it wasreported that the triggering of CD86 molecules activates expression ofnew immunoglobulin genes in the B-cells. These results strongly indicatethat the B7 molecules may provide a direct signal back to the APC. Basedupon these results as well as our data presented above we furtherhypothesized that B7 could provide a direct signal to APC and in turninduce the secretion/expression of certain molecules (cytokines,lymphokines, chemokines, etc), which are important for activation orinhibition of cellular immune responses. Examples of such moleculescould include IL-1α, IL1β, IL-12, and TNF cytokines, which are producedby professional APC and play an important role in immune activation. Infact recently, these cytokines were directly demonstrated to modulatehumoral and cellular anti-viral immune responses during DNAcoimmunization.

Another important observation was that the C-domain of CD80, but notthat of CD86, somehow inhibited costimulation of T-cells. At leastC-domain deleted mutants could significantly increase T-cell activationin our experiments (FIGS. 1, 2A and 2B), whereas the wildtype moleculecould not (Table 4). Recent studies demonstrated a mechanism of T-cellinactivation is mediated through CTLA-4 interactions with a chain of theTCR. Accordingly, our experiments suggest that both V- and C-domains ofCD80 may be essential for CTLA-4 binding and the subsequent inhibitorysignal sent via the TCR overrides simultaneously transmitted CD28activation signals. To directly test this hypothesis animals werecoimmunized with a DNA immunogen and a combination of CD80 and CD86molecules. Coinjection of pCD80 with pCD86 along with the DNA immunogeneliminated the strong anti-viral CD8⁺ CTL response normally seen afterpCD86 costimulation (FIGS. 3, 4). Importantly the C-domain deleted formof CD80 molecule did not generate this inhibitory signal (FIG. 3).Therefore, molecules expressing both V- and C-domains of CD80 not onlyprevented T-cell activation, probably through preferential binding toCTLA-4, but also could overcome the positive signal provided bytriggering of CD28 ligand by V-domains of both CD80 and CD86 molecules.In fact there has been a demonstrated predominance of CTLA-4 signalingover CD28 in which CD28 activation pathways are directly inhibited as aresult of simultaneous CTLA-4 crosslinking

Since the CD80 molecule can bind to both CD28 and CTLA-4 there must beimportant structural differences that shift the equilibrium in the favorof the CTLA-4 inhibition pathway over CD28 activation pathway. Surfaceplasmon resonance analysis measured a fourteen-fold increase in bindingbetween CTLA-4 and CD80 when the C-domain. It is more likely that thisdifference is actually higher, because a multivalent interaction ofCTLA-4 and B7 is likely to occur. The rate of dissociation of theindividual component of CTLA-4 with CD80 is similar to the monomericdissociation. However, since the CD80 will still be held by the secondCTLA-4 interaction, the observed rate of dissociation will be muchslower, thus forming a more stable CTLA-4/CD80 complex. Accordingly ifthe difference in binding avidity of membrane CTLA-4 to membrane CD80 ormembrane CD80 C-domain deleted mutants is calculated, it will be amultiple of 14. The data clearly indicates that the C-domain of CD80 notonly prevents T-cell activation, but also alters structure functionrelationships of this molecule with CTLA-4.

The expression of genes encoding the C-domain deletion mutant of CD80induced not only T-cell activation, but also huge infiltration into themuscle of experimental animals. This infiltration was even greater thanthe infiltration observed after pCD86 and pCEnv coexpression (FIG. 5).Recently, it was reported the APC activity of human muscle cells invitro. After activation with cytokines, human myoblasts can expresscostimulatory molecules and can function as professional MHC class IIrestricted APC. In addition, in vivo effect of muscle cells in T-cellactivation is demonstrated. Mouse muscle cells have been converted toMHC class I restricted APC by the expression of CD86, but not CD80molecules. The data reported herein can be interpreted as showing thatDNA immunization with pcEnv induces a small infiltration ofimmunocompetent cells into the site of injection. These infiltrating Tcells can be activated by transfected muscle cells expressing MHC classI molecules and foreign peptide as well as CD86 molecules. ActivatedT-cells in turn might produce chemokines and attract more T-cells to thesite of inflammation. Accordingly, a huge infiltration is observed inthe muscle tissue in case of coimmunization with pcEnv plus pCD86 (FIG.5). The same mechanism will apply after coimmunization with DNAimmunogen and pV80CAT80. However, coadministration of pCD80 and pcEnvinduced only small infiltration in the muscle tissue of experimentalanimals. If one remembers that CD80 molecule can bind to both CD28 andCTLA-4 there must be forces that shift the equilibrium in the favor ofthe CTLA-4 inhibition pathway over CD28 activation pathway. The presenceof the C-domain of CD80 in muscle cells transfected with wildtype CD80seems to have a very early inhibitory effect on the infiltration and/orproliferation of activated lymphocytes in this tissue and may reflect apreferential binding to and signaling through CTLA-4. Therefore T-cellsin the site of injection will not produce chemokines and will not inducemigration of other lymphocytes in the area of immunization (FIG. 5).Importantly, whether one of the costimulatory molecules preferentiallyfunctions by interaction with CTLA-4 as opposed to CD28 has not beenevaluated and the structural basis for this interaction is stillunclear.

Earlier, a number of studies have reported the results of site directedmutagenesis experiments that investigated the structure functionrelationships of B7 and CD28/CTLA-4 molecules. Collectively thesestudies implicated more than 20 residues on both V and C-domains of CD80critical for CTLA-4 binding. However, the exact region important forinteraction of CD80 molecule to CTLA-4 and CD28 is not defined Spleenand lymph nodes of mice has been shown to have an alternatively splicedform of CD80 that lacks C-domain. This naturally synthesized IgVmolecule binds to CD28=1 g very well (comparable to CD80), although itsbinding to CTLA-4 was suppressed significantly. At least one group alsodemonstrated that deletion of the C-domain of CD80 molecule had agreater effect on binding to CTLA-4=1 g, than CD28=Ig. Importantly,IgV-like region of CD80 could activate proliferation of activatedT-cells in vitro. In vivo data showing effective costimulation providedby the CD80 C-domain deletion molecule support these results. Anyexplanations for the inhibitory nature of costimulatory moleculesexpressing the C-domain of CD80 should consider the increased avidity ofCD80 binding by CTLA-4 as compared to CD28. Recent measurements indicatethat binding avidity of CD80 to dimeric CTLA-4 is much higher than todimeric CD28. Importantly, dimeric CTLA-4 binds to CD80 with greateravidity than to CD86. This greater binding strength may explain why theCTLA-4 induced inhibitory signal predominates in this experimentalsystem. In order to further reveal important differences in CTLA-4binding region of CD80 and CD86 we constructed three-dimensional modelsof both C-domains.

Correlation of mutational studies with three-dimensional models of theC-domain of CD80 provide some insight into receptor binding. Both CD80and CD86 display sequence homology with Ig folds suggesting that bothadopt similar conformations (Table 6). Consequently, most mutationalanalysis have focused on changing conserved residues between human andmouse CD80 and those conserved between CD80 and CD86. Several conservedresidues in the C domain of CD80 appear to be critical for binding toCTLA-4=1 g (Table 6).

A model has established that Q157, D158, E162 and L163 spatially clusterin surface accessible positions near the amino-terminal end of thedomain in the loop between strands D and E and at the beginning ofstrand E. Other residues (F108, P111, and 1113) mapped to strand A (seeTable 6). The second model constructed on the basis of mutationalanalysis also demonstrates a critical role for P135 and P137 in the B-Cloop as well as Q157D158 and P159 in theD-E loop forbinding CTLA-4.Collectively the residues important for receptor binding are mapped tothe ABCED face of the IgC domain.

Table 6 contains: Human CD80 Sequences (SEQ. ID NOS. 13, 15, 17, and 19,top to bottom). Table 6 also contains: Human CD86 Sequences (SEQ. IDNOS. 14, 16, 18 and 20, top to bottom).

FIG. 6 shows Cα traces of CD80 molecule with CTLA-4 binding regionresidues represented as CPK renderings where B-C loop amino acids P135and P136 and D-E loop residues Qi57, D158, P159 are shown. In thecorresponding model of CD86 there is a 4 residues insertion 144-147(KKMS) shown in italic in Table 6 and in FIG. 6. This insertion in CD86changes the conformation of the B-C loop in this molecule in comparisonto CD80, so that the binding region becomes significantly smaller.Because of that insertion, the distance between P159 and P135 decreasesfrom 16 Å in CD80 to 12 Å for the corresponding residues of CD86. Inaddition, the distance between Q157 and P137 decreases from 13 Å in CD80to 10 Å for the corresponding residues of CD86. Therefore, thisinsertion changes the conformation of the B-C loop of the CD86 moleculein comparison to CD80 so that the total surface area of the CTLA-4binding in CD86 molecule is significantly smaller than the same regionof CD80 (Compare FIG. 6). Thus, modeling of CD80 and CD86 suggests thatconformation of B-C loop is different in these molecules and that KKMSinsert in CD86 may play an important role in decreasing of the avidityof binding to CTLA-4 and consequently can decrease the inhibitory signalbeing targeted to the T-cells.

In summary, discrepancies regarding the binding/functional sites of Vand C-domains of CD80 and to a lesser extent of CD86 molecules to CTLA-4and/or CD28 ligands may be explained by different antigens andexperimental systems used in different laboratories. For example manyresults with T-cell costimulation were obtained with anti-CD3 monoclonalantibodies (1-st signal) and soluble forms of B7 (CD80=Ig, CD86=Ig)molecules (2-nd signal). This model generated very important data.However, recent published results demonstrated that only cellsexpressing oligomeric forms of costimulatory molecules can drive T-cellactivation. On the other hand, even usage of CD80 and CD86 transfectedcells for costimulation of T cell may not be considered as an optimalmodel. It was reported that CTLA-4 interacts with TCRζ, after triggeringwith anti-CD3 and membrane-bound CD80 molecules. These results suggestthat proper model probably should include interaction of APC, expressingMHC class I/II, and CD80/CD86 with T-cells, expressing TCR andCD28/CTLA-4. Accordingly, in vivo physiological conditions for APC andT-cell interaction may be more appropriate for uncovering themechanismis of T-cell costimulation. Using this model system, functionaldifferences between CD80 and CD86 as well as between V- and C-domains ofCD80 molecule has been demonstrated.

The current studies provide important information for the development ofnew approaches for the regulation of T-cell immune responses.Specifically, a form of the B7 ligand can be provided to for exampleinclude both V- and C-domains that could inactivate an ongoing humanimmune response probably by preferentially triggering to CTLA-4molecules, expressed on antigen-specific T-cells. Such a construct mayhave important applications for transplantation tolerance or in thetreatment of autoimmune disease. Conversely, more effective tumorvaccination may result from the coimmunization provided through V-domainof CD80 with a decreased ability to inhibit of T-cell activation.Additionally, vaccines that are targeted towards improved cellularimmunity may be provided using molecular adjuvants described herein.

TABLE 1 Picornavirus Family Genera: Rhinoviruses: (Medical) responsiblefor ~50% cases of the common cold. Etheroviruses: (Medical) includespolioviruses, coxsackieviruses, echoviruses, and human enterovirusessuch as hepatitis A virus. Apthoviruses: (Veterinary) these are the footand mouth disease viruses. Target antigens: VP1, VP2, VP3, VP4, VPGCalcivirus Family Genera: Norwalk Group of Viruses: (Medical) theseviruses are an important causative agent of epidemic gastroenteritis.Togavirus Family Genera: Alphaviruses: (Medical and Veterinary) examplesinclude Senilis viruses, RossRiver virus and Eastern & Western Equineencephalitis. Reovirus: (Medical) Rubella virus. Flariviridue FamilyExamples include: (Medical) dengue, yellow fever, Japanese encephalitis,St. Louis encephalitis and tick borne encephalitis viruses. Hepatitis CVirus: (Medical) these viruses are not placed in a family yet but arebelieved to be either a togavirus or a flavivirus. Most similarity iswith togavirus family. Coronavirus Family: (Medical and Veterinary)Infectious bronchitis virus (poultry) Porcine transmissiblegastroenteric virus (pig) Porcine hemagglutinating encephalomyelitisvirus (pig) Feline infectious peritonitis virus (cats) Feline entericcoronavirus (cat) Canine coronavirus (dog) The human respiratorycoronaviruses cause ~40 cases of common cold. EX. 224E, 0C43 Note -coronaviruses may cause non-A, B or C hepatitis Target antigens: E1 -also called M or matrix protein E2 - also called S or Spike protein E3 -also called HE or hemagglutin-elterose glycoprotein (not present in allcoronaviruses) N - nucleocapsid Rhabdovirus Family Genera: VesiliovirusLyssavirus: (medical and veterinary) rabies Target antigen: G protein Nprotein Filoviridue Family: (Medical) Hemorrhagic fever viruses such asMarburg and Ebola virus Paramyxovirus Family: Genera: Paramyxovirus:(Medical and Veterinary) Mumps virus, New Castle disease virus(important pathogen in chickens) Morbillivirus: (Medical and Veterinary)Measles, canine distemper Pneuminvirus: (Medical and Veterinary)Respiratory syncytial virus Orthomyxovirus Family (Medical) TheInfluenza virus Bungavirus Family Genera: Bungavirus: (Medical)California encephalitis, LA Crosse Phlebovirus: (Medical) Rift ValleyFever Hantavirus: Puremala is a hemahagin fever virus Nairvirus(Veterinary) Nairobi sheep disease Also many unassigned bungavirusesArenavirus Family (Medical) LCM, Lassa fever virus Reovirus FamilyGenera: Reovirus: a possible human pathogen Rotavirus: acutegastroenteritis in children Orbiviruses: (Medical and Veterinary)Colorado Tick fever, Lebombo (humans) equine encephalosis, blue tongueRetrovirus Family Sub-Family: Oncorivirinal: (Veterinary) (Medical)feline leukemia virus, HTLVI and HTLVII Lentivirinal: (Medical andVeterinary) HIV, feline immunodeficiency virus, equine infections,anemia virus Spumavirinal Papovavirus Family Sub-Family: Polyomaviruses:(Medical) BKU and JCU viruses Sub-Family: Papillomavirus: (Medical) manyviral types associated with cancers or malignant progression ofpapilloma Adenovirus (Medical) EX AD7, ARD., O.B. - cause respiratorydisease - some adenoviruses such as 275 cause enteritis ParvovirusFamily (Veterinary) Feline parvovirus: causes feline enteritis Felinepanleucopeniavirus Canine parvovirus Porcine parvovirus HerpesvirusFamily Sub-Family: alphaherpesviridue Genera: Simplexvirus (Medical)HSVI, HSVII Varicellovirus: (Medical - Veterinary) pseudorabies -varicella zoster Sub-Family - betaherpesviridue Genera: Cytomegalovirus(Medical) HCMV Muromegalovirus Sub-Family: Gammaherpesviridue Genera:Lymphocryptovirus (Medical) EBV - (Burkitts lympho) RhadinovirusPoxvirus Family Sub-Family: Chordopoxviridue (Medical - Veterinary)Genera: Variola (Smallpox) Vaccinia (Cowpox) Parapoxivirus - VeterinaryAuipoxvirus - Veterinary Capripoxvirus Leporipoxvirus SuipoxvirusSub-Family: Entemopoxviridue Hepadnavirus Family Hepatitis B virusUnclassified Hepatitis delta virus

TABLE 2 Bacterial pathogens Pathogenic gram-positive cocci include:pneumococcal; staphylococcal; and streptococcal. Pathogenicgram-negative cocci include: meningococcal; and gonococcal. Pathogenicenteric gram-negative bacilli include: enterobacteriaceae; pseudomonas,acinetobacteria and eikenella; melioidosis; salmonella; shigellosis;hemophilus; chancroid; brucellosis; tularemia; yersinia (pasteurella);streptobacillus moniliformis and spirillum ; listeria monocytogenes;erysipelothrix rhusiopathiae; diphtheria; cholera; anthrax; donovanosis(granuloma inguinale); and bartonellosis. Pathogenic anaerobic bacteriainclude: tetanus; botulism; other clostridia; tuberculosis; leprosy; andother mycobacteria. Pathogenic spirochetal diseases include: syphilis;treponematoses: yaws, pinta and endemic syphilis; and leptospirosis.Other infections caused by higher pathogen bacteria and pathogenic fungiinclude: actinomycosis; nocardiosis; cryptococcosis, blastomycosis,histoplasmosis and coccidioidomycosis; candidiasis, aspergillosis, andmucormycosis; sporotrichosis; paracoccidiodomycosis, petriellidiosis,torulopsosis, mycetoma and chromomycosis; and dermatophytosis.Rickettsial infections include rickettsial and rickettsioses. Examplesof mycoplasma and chlamydial infections include: mycoplasma pneumoniae;lymphogranuloma venereum; psittacosis; and perinatal chlamydialinfections. Pathogenic eukaryotes Pathogenic protozoans and helminthsand infections thereby include: amebiasis; malaria; leishmaniasis;trypanosomiasis; toxoplasmosis; pneumocystis carinii; babesiosis;giardiasis; trichinosis; filariasis; schistosomiasis; nematodes;trematodes or flukes; and cestode (tapeworm) infections.

TABLE 3 Expression efficiency of constructs encoding wildtype, chimeric,or truncated forms of CD80 and CD86 molecules detected by FACS assay.Schematic Representation Description of Plasmids of Ig Domains* encodedprotein FI** Vector control No Insert 2.5 pCD80

Wildtype CD80 8.5 pV80CΔT80

Truncated CD80, C domain deletion 6.33 pV80C86T86

Chimeric, V domain of CD 86 substituted by V domain of CD80 5.9pV80C80TΔ

Truncated CD80, cytoplasmic (T) domain deletion 5.8 pCD86

Wildtype CD86 5.99 pV86CΔT86

Truncated CD86, C domain deletion 7.1 pV86C80T80

Chimeric, V domain of CD 80 substituted by V domain of CD86 7.4pV86C86TΔ

Truncated CD86,cytoplasmic (T) domain deletion 11.16 *Boxes represent V,C and T (cytoplasmic tail) domains of CD80 (shaded) and CD86 (open)molecules (transmembrane sequence was not removed. **Rhabdomyosarcomacells were cotransfected with experimental or control plasmids and aconstruct encoding the green fluorescence protein (GFP). FluorescenceIntensity (FI) of cells expressing B7 molecules (red mean channel) wasdetected in the population of cells expressing GFP.

TABLE 4 Cellular immune responses (CTL and Th1 cytokine production) inmice coimmunized with plasmids, encoding viral antigen and differentforms of B7 molecules. * Anti-viral CTL (%) * Th1 cytokines Miceimmunized with at different E:T ratio (pg/ml) the following plasmids100:1 50:1 25:1 γIFN IL-12 Naive 1.1 2.5 4.3 34.50 16.80 pcEnv 13.3 12.813.7 46.50 31.37 pcEnv + pcD80 16.6 12.5 11.3 36.32 36.99 pcEnv + pcD8647.7 34.8 24.7 334.54 71.86 pcEnv + pV80C86T86 42.8 36.2 28.5 500.3790.32 pcEnv + pV86C80T80 16.4 17.1 13.4 50.93 26.42 pcEnv + pV80C80TΔ14.5 8.6 5.1 41.25 21.87 pcEnv + pV86C86TΔ 11.0 8.7 10.6 38.91 31.86 *Two weeks after the last immunization spleens were collected and CTL andTh1 cytokine assays were performed as described in Materials andMethods. These experiments have been repeated three (for detection ofCTL) and two (for detection of cytokines) times with similar results.

TABLE 5 Binding parameters for CTLA-4/CD80 interaction estimated fromfits to a Langmuir model CD80 type k_(on) (l/Ms) k_(off) (l/s) K_(D) (M)Wildtype 3.99 × 10⁵ 2.00 × 10⁻⁴ 5.02 × 10⁻¹⁰ CD80 C-domain deleted 7.86× 10⁴ 5.53 × 10⁻⁴ 7.04 × 10⁻⁹  CD80

TABLE 6 Sequence alignment of C-domains of human CD80 and CD86.

* Alignments were done using CLUSTALW (Thompson, 1994) and then adjustedmanually. Residues at the end of each row are numbered from theirrespective N-terminus. Residues of CD80 critical for binding T cellsurface receptor CTLA-4 according to literature (Ellis, 1996; Fargeas,1995; Guo, 1995; Guo, 1998; Peach, 1995) are shown in bold face. Twopresumable variants of four amino acids insert in CD86 are shown initalic and more likely could disturb binding to CTLA-4 (see details indiscussion). Highlighted beta strands in both CD80 and CD86 based on thecrystal structure of sB7-l (Ikemizu, 2000).

1. A nucleic acid molecule that comprises a coding sequence operablylinked to regulatory elements, wherein said coding sequence that encodesa human CD80 mutant protein comprises at least one of 80V, 80tm and 80ctand is free of all or part of the CD80 C region; wherein said CD80mutant that is free of all or part of the CD80 C region comprises either80V or 86ct or both and optionally comprises one or more of 86C, 80tm,86tm, 80ct and 86ct wherein: 80V is the variable domain of human CD80;86V is the variable domain of a human CD86; 86C is the C domain of humanCD86; 80tm is the transmembrane region of human CD80; 86tm is thetransmembrane region of a human CD86; 80ct is the cytoplasmic tail ofhuman CD80; 86ct is the cytoplasmic tail of human CD86; wherein saidhuman CD80 mutant protein possesses costimulatory activity of wild typeCD80 and does not provide the negative signal associated with wild typehuman CD80 C region interactions with human CTLA4.
 2. A plasmidcomprising a nucleic molecule according to claim
 1. 3. A plasmidaccording to claim 2 further comprising a coding sequence encoding animmunogen, said coding sequence operably linked to regulatory elements.4. A composition comprising a plasmid according to claim 2 furthercomprising an immunogenic protein or a plasmid comprising a nucleic acidsequence comprising a coding sequence encoding an immunogen, said codingsequence operably liked to regulatory elements.
 5. A compositioncomprising a plasmid according to claim 3 further comprising animmunogenic protein or a plasmid comprising a nucleic acid sequencecomprising a coding sequence encoding an immunogen, said coding sequenceoperably liked to regulatory elements.
 6. The nucleic acid moleculeaccording to claim 1 wherein said protein comprises part of human CD80 Cregion.
 7. A plasmid comprising a nucleic acid molecule according toclaim
 6. 8. A plasmid according to claim 7 further comprising a codingsequence encoding an immunogen, said coding sequence operably linked toregulatory elements.
 9. A composition comprising a plasmid according toclaim 8 further comprising an immunogenic protein or a plasmidcomprising a nucleic acid sequence comprising a coding sequence encodingan immunogen, said coding sequence operably liked to regulatoryelements.
 10. A composition comprising a plasmid according to claim 7further comprising an immunogenic protein or a plasmid comprising anucleic acid sequence comprising a coding sequence encoding animmunogen, said coding sequence operably liked to regulatory elements.11. The nucleic acid molecule according to claim 1 that comprises acoding sequence operably linked to regulatory elements, wherein saidcoding sequence encodes a human CD80 mutant protein that comprises ahuman CD80 V region, a functional human CD80 tm and a human CD80 ctregion, and is free of a functional C region by the absence of all orpart of the a human CD80 C region.
 12. A plasmid comprising a nucleicmolecule according to claim
 11. 13. A plasmid according to claim 12further comprising a coding sequence encoding an immunogen, said codingsequence operably linked to regulatory elements.
 14. A compositioncomprising a plasmid according to claim 13 further comprising animmunogenic protein or a plasmid comprising a nucleic acid sequencecomprising a coding sequence encoding an immunogen, said coding sequenceoperably liked to regulatory elements.
 15. A composition comprising aplasmid according to claim 12 further comprising an immunogenic proteinor a plasmid comprising a nucleic acid sequence comprising a codingsequence encoding an immunogen, said coding sequence operably liked toregulatory elements.
 16. The nucleic acid according to claim 11 whereinsaid coding sequence encodes part of the CD80 C region.
 17. A plasmidcomprising a nucleic molecule according to claim
 16. 18. A plasmid ofclaim 17 further comprising a coding sequence encoding an immunogen,said coding sequence operably linked to regulatory elements.
 19. Acomposition comprising a plasmid according to claim 18 furthercomprising an immunogenic protein or a plasmid comprising a nucleic acidsequence comprising a coding sequence encoding an immunogen, said codingsequence operably liked to regulatory elements.
 20. A compositioncomprising a plasmid according to claim 17 further comprising animmunogenic protein or a plasmid comprising a nucleic acid sequencecomprising a coding sequence encoding an immunogen, said coding sequenceoperably liked to regulatory elements.